Introduction
Introduction
Metastasis is a major hurdle in the successful treatment of solid tumors, including those of the breast. Estrogens are the steroid hormones that regulate the growth, differentiation and function of target tissues of the reproductive tract, central nervous system, skeletal system, cardiovascular system and immune system21-26.
Aim and scope of the work
However, the exact mechanism of estrogen-mediated regulation of CSTA in breast cancer cells is not understood. However, the underlying mechanism for the loss of CSTA expression in breast tumors is unknown.
Objectives
In the previous chapter, estrogen-mediated suppression of CSTA expression in breast cancer cells was demonstrated. The distribution of CSTA expression (log2(RPKM+1)) in hypomethylated and hypermethylated tumors is presented as a box plot (F).
Review of literature
Breast cancer
Pie charts representing the estimated number of breast cancer cases among women of all ages (left panel) and the estimated number of breast cancer-related deaths among women of all ages (right panel) worldwide . The area of the circles shows the percentage of breast cancer cases (A) or deaths (B) in each country.
Classification
Over the past 60 years, the TNM system has gradually evolved due to advances in diagnosis and treatment. Furthermore, it is also being investigated whether non-anatomical prognostic factors can be included in the TNM system to provide the most specific treatment to the patients62.
Risk factors
The histological classification system is used to classify malignant tumors based on the degree of differentiation. NHG scores tumors from 1 to 3 for each of three morphologic features: tube formation, nuclear pleomorphism, and mitotic activity.
Treatment
Endocrine therapy has its origins back in 1895, when oophorectomy performed by Beatson in premenopausal breast cancer patients resulted in complete remission of breast tumors and prolonged survival77. A number of studies in the following decades of estrogen signaling revealed the potential of endocrine therapy as a treatment option27,79-86.
Estrogen
Estrogen receptors
The differential response of ERα and ERβ to the same ligands is attributed to the difference in the AF-1 domain107,112. Overexpression of ERα46 in MCF-7 cells inhibits the ERα66-mediated estrogenic induction of c-fos and cyclin D1.
Estrogen signaling
- Genomic pathway
- Non-genomic pathway
In addition to the classic estrogen receptors, truncated variants of ERα66 (namely ERα46, ERα36) are also known. This study suggested that ERα46 partially antagonizes the proliferative effect of ERα66 in MCF-7 cells by inhibiting ERα66 AF-1 activity125.
Endocrine resistance
In vitro studies have reported the frequent occurrence of deletion or point mutation in ER gene of tamoxifen-resistant MCF-7 and T47D breast cancer cells. Interestingly, in vitro study showed that the development of tamoxifen resistance in MCF-7 cells is accompanied by transition to more aggressive phenotype143.
Metastasis
Sequential events in metastasis are- i) proliferation of tumor cells in the primary site, ii) local invasion of detached cells, iii) intravasation into blood capillaries, iv) arrest and extravasation to the secondary site, and v) formation of a secondary tumor (regenerated from Arthritis and colleagues, 2008150).
Cathepsins
However, the observed extracellular activity of cysteine cathepsins indicates that pH is not the sole determinant of their activity6. Cathepsins are naturally synthesized as inactive precursors and activated by propeptide removal by other proteases or by autocatalytic removal at an acidic pH of 156.
Cystatins
Cystatins are the endogenous competitive, reversible inhibitors that prevent the binding of substrate to the active site of cathepsins39,157. Cystatins form a 'tripartite wedge' and bind to the active-site groove of cathepsins, thereby blocking the access of collagen to the cathepsins.
Cathepsins and cystatins in cancer
Coculture of cathepsin K-negative breast cancer cells with cathepsin K-expressing fibroblasts promoted invasion, and this effect was abolished by cathepsin K180 inhibitors. Downregulation of cathepsin L delayed tumor migration and invasion by inhibiting transforming growth factor-β (TGF-β)-mediated epithelial-mesenchymal transition (EMT) 181 .
Cystatin A (CSTA)
This unraveled the role of CSTA in desmosome-mediated cell-cell adhesion in the lower levels of the epidermis40. Furthermore, Scott and colleagues reported that Csta1 and Csta3 are more highly expressed in the skin of neonatal mice than in adult skin, and that expression decreases with age, suggesting their role in epidermal development212.
CSTA in cancer
Recently, Ma and colleagues reported that overexpression of CSTA in squamous cell carcinoma cells enhances gemcitabine-induced apoptosis. Levicar and colleagues reported 1.9-fold higher levels of CSTA in cytosols of primary invasive breast tumors compared with normal breast parenchyma.
Regulation of CSTA expression
However, in breast cancer, studies trying to understand the exact role of CSTA are limited. Further analysis showed that AP-2γ binds to the AP-2 binding site (-75 to -84) of CSTA and induces its expression233.
Epigenetic regulation
- DNA methylation
- Epigenetic regulation of cystatins in cancer
This study reveals the essential role of ERα in estrogen-mediated suppression of CSTA expression in breast cancer cells. Taken together, this work provides new insights into the regulation of CSTA expression in breast cancer.
Materials and methods
Plasticware, chemicals, and reagents
ProteoGuardTM EDTA-free protease inhibitor cocktail and pMD20 vector were purchased from Clontech (CA, USA). Monoclonal CSTA antibody (ab10442) was from Abcam (Cambridge, UK) and polyclonal ERα antibody (sc-543) was purchased from Santa Cruz Biotechnology (CA, USA). Polyclonal histone H3 antibody (BB-AB0055) and normal rabbit immunoglobin G (IgG) antibody (BB-AB0001) were purchased from BioBharati LifeScience Pvt.
Cell culture and treatments
- Cell lines and cell culture
- Sub-culturing and seeding
- Treatment protocols
Used M2 medium was removed and replaced with fresh M2 medium containing the indicated concentration of E2 or ethanol (vehicle control) and incubated at 37 °C for 72 h. MCF-7 cells were grown in M1 medium and when the cells were 70% confluent, M1 medium was replaced with M2 medium. MCF-7 cells were grown in M1 medium and when the cells were 60% confluent, M1 medium was replaced with M2 medium.
Gene expression analysis
- Primers
- Total RNA isolation and cDNA synthesis
- Routine RT-PCR
- qRT-PCR
The normalized band intensities obtained for controls were assigned the value of 1 and those obtained for treatments were expressed relative to control. Reactions were set up using AmpliTaq Gold PCR master mix (supplemented with 0.6X SYBR Green) or Powerup SYBR Green PCR master mix. The expression levels of each gene in test samples relative to control were analyzed by the ΔΔCt method252.
Generation of polyclonal CSTA antibody
Diluted hyperimmune serum was passed through the column and incubated for 4 hours at room temperature. The next day, plates were incubated for 2 hours at room temperature, followed by washing with PBS containing 0.05% Tween 20 (PBST). The wells were further washed and incubated with 100 µl horseradish peroxidase (HRP) conjugated secondary antibody (1:5000) for 1 hour at room temperature.
Western blotting
Indirect enzyme-linked immunosorbent assay (ELISA) was performed with immune sera and affinity-purified antibody to check reactivity. CSTA peptide (200 ng/well) was coated in a 96-well plate for 2 h at room temperature, followed by overnight incubation at 4 ºC. Preimmune serum, hyperimmune serum, or peptide affinity-purified antibody were diluted (1:5000) with 1% skim milk.
Chromatin immunoprecipitation (ChIP)
ChIP-Seq analysis
Bisulfite sequencing
Cloning of CSTA ORF in mammalian expression vector
Establishment of stable cell lines
Functional assays
- MTT assay
- Scratch wound healing assay
- Transwell invasion assay
The stained cells were imaged in the microscope, and then crystal violet dye was dissolved in 1% SDS solution at 25 ºC for 10 min.
Survival analysis
TCGA data analysis
- Analysis of CSTA expression in normal breast tissues and primary breast tumors
- Analysis of CSTA expression in different subtypes and stages of breast tumors
- Analysis of association between CSTA expression and histopathological parameters
- Expression-methylation correlation (EMC) analysis
The difference in CSTA expression in these two groups was analyzed by Welch two-sample t-test and the expression data were represented as box plots. To evaluate the association of CSTA expression with histopathological parameters, tumors were classified as CSTA-high and CSTA-low using median value as cutoff and analyzed by non-parametric chi-square test in Microsoft Excel. The difference in CSTA expression in these two groups was analyzed by Welch two-sample t-test.
Statistical analysis
The likely effect of DNA methylation in the silencing of CSTA in breast cancer cells has not yet been investigated. This suggested that low or absent CSTA expression in breast cancer cells could be a result of DNA methylation at the CSTA locus. Estrogen-mediated repression of CSTA expression in breast cancer cells occurs by binding of ERα to the intron-2 region of CSTA.
CSTA expression in breast cancer
Introduction
On the contrary, based on immunohistochemical analysis, Kuopio and colleagues demonstrated an association between CSTA expression and an aggressive phenotype. Levicar and colleagues reported a 1.9-fold higher concentration of CSTA in cytosols of primary invasive breast tumors compared to normal breast parenchyma32. However, to date, no research has investigated the association of CSTA with these molecular markers of breast cancer.
Results
- Association of CSTA expression with breast cancer prognosis
- Analysis of CSTA expression in normal breast tissues and primary breast tumors
- Analysis of CSTA expression in molecular subtypes and stages of breast tumors
- Association of CSTA expression with histopathological parameters
Box plots showing the distribution of CSTA mRNA expression in normal breast tissues and breast tumors. The distribution of CSTA and ERα expression in the molecular subtypes is shown as box plots in Figure 4.4. Box plots showing the distribution of CSTA (A) and ERα (B) mRNA expression in the indicated subtypes of primary breast tumors.
Discussion
In this study, the mechanism of E2-mediated suppression of CSTA in breast cancer cells was investigated. Taken together, the present study demonstrates that CSTA expression in breast cancer cells is inversely related to DNA methylation at the CSTA locus. Overexpression of CSTA in breast cancer cells reduced migration and invasion of breast cancer cells without affecting proliferation.
Estrogen-mediated regulation of CSTA expression in breast cancer
Introduction
Results
- E2 suppresses CSTA expression in MCF-7 breast cancer cells
- E2-mediated suppression of CSTA expression in MCF-7 cells involves ERα
- Estrogen enhances ERα occupancy in the intron-2 region of CSTA in MCF-7 cells
- Regulation of CSTA expression by estrogen in other breast cancer cell lines
PPT significantly suppressed CSTA expression to the same extent as E2 confirming that ERα is involved in E2-mediated CSTA suppression (Figure 5.2A, B). Further, the involvement of ERα in E2-mediated suppression of CSTA was assessed using tamoxifen (SERM) and fulvestrant (SERD). A strong peak of ERα occupancy was observed in the intron-2 region of CSTA in MCF-7 cells treated with E2 (indicated by the red rectangle, Figure 5.5B).
Discussion
In general, methylation in both regions appeared to be inversely related to CSTA expression in breast cancer cells. Taken together, these data provide compelling evidence in favor of DNA methylation-dependent silencing of CSTA in breast cancer cells. Estradiol-estrogen receptor: a key interplay between the expression of syndecan-2 and metalloproteinase-9 in breast cancer cells.
DNA methylation-dependent expression and regulation of CSTA
Introduction
Results
- Differential expression of CSTA in breast cancer cell lines
- In silico analysis of DNA methylation in the CSTA locus
- Bisulfite sequencing of upstream and intron-2 regions in the CSTA locus
- Upstream CpGs methylation inversely correlates with CSTA expression in breast
- Intron-2 region of CSTA encompasses a potential ERE
- Global demethylation restores estrogen regulation of CSTA in MDA-MB-231 and
The likely reason behind the absence of E2-mediated regulation of CSTA expression in T47D cells is addressed in the next chapter. However, the mechanisms leading to the loss of CSTA expression in breast tumors are not known. Interestingly, estrogen regulation via ERα and DNA methylation-dependent silencing converge in the intron-2 of CSTA.
Discussion
Primary tumor expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer. Influence of estrogen receptor β on gene networks regulated by estrogen receptor α in breast cancer cells. Estrogen receptor gene CpG island methylation marks loss of estrogen receptor expression in human breast cancer cells.
Functional role of CSTA in breast cancer
Introduction
Results
- Cloning of CSTA ORF into mammalian expression vector
- Generation of MDA-MB-231 cells stably expressing CSTA
- CSTA expression reduces the migration of MDA-MB-231 cells
- CSTA expression reduces the invasion of MDA-MB-231 cells
- Effect of CSTA expression on EMT markers
- Effect of CSTA knockdown on EMT markers
Global demethylation in MDA-MB-231 cells established functional ERα (as revealed by PR induction after estrogen stimulation) and CSTA mRNA expression (Figure 6.7A, B). The effect of CSTA expression on the migration of MDA-MB-231 cells was assessed by scratch wound assay. This indicates the potential of CSTA in reducing the invasion of MDA-MB-231 cells (Figure 7.5A, B).
Discussion
However, the scant literature presents conflicting views on the role of the first member of the cystatin superfamily, CSTA, in breast cancer. Identification of a gene (GPR30) with homology to the G protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer.
Conclusion
To further confirm specificity, the blot was pre-blocked with peptide prior to probing with affinity-purified antibody. When the antibody was pre-blocked with CSTA peptide, purified CSTA protein was not detected. MCF-7 and MDA-MB-231 total proteins (A & B) and CSTA-purified proteins (C) were probed with preimmune serum, immune serum (Bleed 3) rabbit A, affinity-purified antibody, or affinity-blocked peptide-purified antibody.
Dixcy Jaba Sheeba John Mary, Girija Sikarwar, Mohan C Manjegowda, Ajay Kumar, Anil Mukund Limaye, Regulation of cystatin A by estrogen and DNA methylation in breast cancer cells Indian Association for Cancer Research (IACR) Annual Conference-2020 (February 5- 7, 2020) at Kovalam, Trivandrum (OP-5). Limaye, Study of Steady State Regulation of Cystatin A mRNA Level by Estrogen in Breast Cancer Cells Research Conclave (March 18-20, 2016) at IIT Guwahati, Assam. Limaye, Regulation of Cystatin A by Estrogen in Breast Cancer Cells in International Conference on Cancer Research: New Horizons 2015 (19-21 November 2015) at NCCS, Pune, P-23; Page No.-77.
