Background
Cathepsin V (CTSV) is a human lysosomal cysteine protease, with normal physiological expression primarily found within the thymus, corneal epithelium and testis [
1‐
3]. CTSV resides at human chromosome 9q22, the same loci as closely related family member, cathepsin L (CTSL) [
3]. Studies have suggested that CTSV evolved from CTSL after mammalian divergence due to their high homology, but differences in their tissue distribution and active site morphology suggests that CTSV exerts distinct functions [
4,
5]. CTSV has been shown to exhibit potent elastase activity [
6], and its proteolytic activity has also been shown to be important for invariant chain processing in MHC-II antigen presentation within the thymus [
7].
Whilst CTSV has not had the same extensive research to date as other cathepsin family members, there is increasing evidence associating this protease with multiple malignancies. Elevated expression of CTSV in tumour compared to normal tissue has been observed for squamous cell carcinoma, breast, colorectal and thymic epithelial cancers [
2,
8‐
10], whereas more in depth analysis in hepatocellular and endometrial carcinomas has indicated that elevated CTSV expression is also associated with increasing tumour grade or stage [
11,
12].
In relation to breast cancer, previous studies have also shown that CTSV expression is associated with distant metastasis [
9]. Whilst these initial breast cancer studies did not clarify the particular subtype within which CTSV expression was elevated, CTSV has previously been associated with ER-positive breast cancers due to its inclusion on the Oncotype DX® genomic test. CTSV is one of 16 cancer related genes on the Oncotype DX® array which is used to assess the risk of ER-positive breast cancer recurrence and to determine the benefits of adjuvant chemotherapy treatment [
13]. Further analysis of the Oncotype DX® gene signature by assessing copy number variation identified that CTSV was significantly associated with both reduced overall survival and disease-free survival in a cohort of breast cancer patients [
14]. More recently, CTSV expression was also found to be elevated in breast ductal carcinoma in situ (DCIS), where it is associated with a poor outcome and has potential to predict DCIS progression to invasive disease [
15].
GATA3 is a member of the zinc finger transcription factor family and has been shown to be essential for normal mammary gland development, with roles in essential processes such as luminal cell differentiation, adhesion and proliferation [
16,
17]. Murine studies have shown that GATA3 expression is lost as luminal epithelial cells become less differentiated during breast cancer progression [
18] and low GATA3 expression has been strongly associated with histological grade and positive lymph nodes, both of which are indicators of poor prognosis [
19,
20].
GATA3 expression is highly correlated with ER-positive breast cancers and is associated with a favourable clinical outcome. In particular, highest expression levels of GATA3 have been observed in the luminal A subtype of ER-positive tumours [
19,
21]. Numerous reports have detailed that loss of GATA3 expression is associated with a propensity for tumour metastasis, with mechanistic studies suggesting that GATA3 inhibits breast cancer metastasis by reversing epithelial-mesenchymal transition (EMT) [
22,
23].
In this study, we investigate the role of CTSV in ER-positive breast cancer to determine the molecular mechanisms by which this protease contributes to tumourigenesis. We report that CTSV expression is associated with poor prognosis in breast cancer, particularly within the ER-positive subtype. Examination of CTSV shRNA cell line models reveals that CTSV facilitates proliferation and invasion of ER-positive breast cancer cells. We also show that CTSV can regulate the expression of GATA3 in ER-positive breast cancer cell lines. Collectively, these results indicate that CTSV is a prospective therapeutic target in ER-positive breast cancer.
Methods
Cell line culture and treatments
The human breast cancer cell lines MCF-7 and ZR-75-1 and 293 T human embryonic kidney cells were obtained from the American Type Culture Collection (ATCC). MCF-7 and 293 T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 50 U/mL penicillin and 50 μg/ml streptomycin. ZR-75-1 cells were cultured in Roswell Park Memorial Institute-1640 media (RPMI-1640) supplemented with 1% sodium pyruvate, 10% fetal calf serum and 50 U/mL penicillin and 50 μg/ml streptomycin. All cells were grown at 37 °C in a humidified incubator with 5% CO2 and media were changed every 3 days. All cell culture media, supplements and antibiotics were purchased from Thermo Fisher Scientific. Hoechst 33258 (50 ng/mL) and actinomycin-D (5 μg/μl) were purchased from Thermo Fisher Scientific, whilst Bortezomib (100 nM) and Buparlisib (5 μM) were purchased from Insight Biotechnology. All were used according to manufacturer’s instructions.
Lentiviral cell line generation and transient transfections
Lentiviral cell line generation was undertaken as previously described [
24]. Briefly, 293 T cells were transfected with pLKO.1-shCTSV plasmids (Sigma-Aldrich), after which viral supernatant was harvested and used to transduce MCF-7 and ZR-75-1 cells. For transient transfections, cells were transfected using GeneJuice® transfection reagent in accordance to manufacturer’s instructions. pcDNA3.1-CTSV ORF (GeneScript), pCMV-Flag-STYX (MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee) or empty vector control plasmids were transfected and incubated for 48 h before protein extraction and analysis. The catalytic cysteine residue of pcDNA3.1-CTSV ORF was mutated to a serine reside using site-directed mutagenesis (Agilent Technologies) to give rise to the mutant construct (mtCTSV). The shRNA-resistant plasmid (rCTSV) was also generated using site-directed mutagenesis of pcDNA3.1-CTSV ORF, with the targeting sequence of shCTSV-1 mutated to alter codon usage but still produce the same protein sequence.
Western blotting
Whole cell lysates were prepared using RIPA buffer with quantification using the Pierce™ BCA Protein assay kit (ThermoFisher). Lysates were resolved by SDS-PAGE using 10% acrylamide gels and proteins were transferred onto polyvinylidene fluoride (PVDF) membrane by semi-dry blotting. The following antibodies were used in this study: goat polyclonal CTSV (BioTechne, AF1080), goat polyclonal GAPDH (BioTechne, AF5718), mouse monoclonal GATA3 (BioTechne, MAB6330), rabbit monoclonal pSer308 GATA3 (Abcam, ab186371), mouse monoclonal pThr308 Akt (BioTechne, MAB7419), rabbit polyclonal pSer473 Akt (Cell Signaling, 9271), mouse monoclonal Akt (BD Transduction Laboratories, P78020), rabbit polyclonal pSer9 GSK-3β (BioTechne, AF1590), mouse monoclonal GSK-3β (BioTechne, MAB25063) and mouse monoclonal FLAG M2 (Sigma-Aldrich, F3165). Secondary antibodies used were donkey anti-goat HRP conjugate (BioTechne, HAF109), goat anti-mouse HRP conjugate (BioRad, 172-1011) and goat anti-rabbit HRP conjugate (Cell Signaling, 70745). Protein visualisation was undertaken using Luminata™ Forte chemiluminescent detection reagent (Merck Millipore) and imaged using a Molecular Imager ChemiDoc XRS+ Imaging System (BioRad).
RQ-PCR
RNA was extracted using Stat-60 (Amsbio) and cDNA prepared using the Transcriptor First Strand cDNA synthesis kit (Roche) according to manufacturer’s instructions. RQ-PCR was undertaken using the Lightcycler® 480 SYBR Green I Master reagent (Roche). Primer sequences used were as follows: CTSV-F: 5′-ggactctgaggaatcctatccat-3′, CTSV-R: 5′-gcaacagaattctcaggtctgtact-3′, GATA3-F: 5′-ctcattaagccccaagcgaag-3′, GATA3-R: 5′-tctgacagttcgcacaggac-3′, β-tubulin-F: 5′-cgcagaagaggaggaggatt-3′, β-tubulin-R: 5′-gaggaaaggggcagttgagt-3′. RQ-PCR was performed in duplicate for each gene investigated, with all experiments undertaken a minimum of 3 times. The fold change of mRNA expression was calculated by normalising absolute target gene expression levels to β-tubulin mRNA levels as an internal control. The average and standard deviation (SD) values were collated from three independent experiments and statistical analysis was determined by one-way-ANOVA using GraphPad Prism 8.
Clonogenic assays
Clonogenic assays were performed by seeding optimised cell numbers in 6 well plates in triplicate and incubated at 37 °C in a humidified incubator with 5% CO
2 for 11–13 days (cell-line dependent) to enable colony formation. Cells were fixed and stained with 0.4% crystal violet and the number of colonies containing at least 50 cells was determined in line with published protocols [
25]. All assays were carried out in triplicate and colonies were counted using the Cell3 iMager neo. Results demonstrate the mean colony number per cell line ± SD. The average and SD values were collated from three independent experiments and statistical analysis was determined by one-way-ANOVA using GraphPad Prism 8.
Invasion assays
Transwell inserts (Corning, UK) containing 8.0 μm polycarbonate membrane was coated with Matrigel (1 mg/mL) (BD Biosciences). Cells were seeded into the upper chamber in serum-free media, with growth media containing 10% FBS added to the lower chamber as a chemoattractant. Cells were incubated at 37 °C in a humidified incubator with 5% CO2 for 48 h. Invaded cells were fixed using Carnoy’s fixative and visualised by staining with Hoechst 33258 (50 ng/mL) prior to images being captured on a Leica DM5500 fluorescent microscope. Each cell line was analysed in triplicate with 9 images captured per membrane at × 20 magnification. Images were analysed using ImageJ and figures were generated using GraphPad Prism 8. The average and SD values representing the mean number of cells per field of view were collated from three independent experiments and statistical analysis was determined by one-way-ANOVA using GraphPad Prism 8.
Extracellular ATP was quantified by CellTiter-Glo® assay (Promega). Cells were seeded for 48 h before supernatants were collected for analysis. Fifty microliters of supernatants was added in triplicate to 96-well white tissue culture-treated plates (Corning) and mixed with 50 μL of CellTiter-Glo® reagent. Plates were agitated on a shaker in the dark for 5 min at 37 °C before luminescence was measured using a BioTek Synergy HT plate reader. Results were presented as the mean relative fold change and SD from three independent experiments, with statistical analysis determined by one-way-ANOVA using GraphPad Prism 8.
Survival analysis
Online repository KM plotter (
www.kmplot.com) was used to evaluate the correlation between CTSV mRNA expression and relapse free survival (RFS) [
26]. Using the breast cancer dataset, survival dependent on CTSV expression was analysed based on ER status and intrinsic patient subtype (
N = 3951) using a collation of previously published and publicly available Affymetrix microarray datasets, available through GEO, European Bioinformatics Institute and TCGA. Gene expression was evaluated using CTSV probe 210074_at, with patients split by best performing threshold and all data right censored at 120 months (10 years). Survival plots were presented as percentage survival versus time in months, with hazard ratio (HR), 95% confidence interval (CI) and log-rank
p values calculated using GraphPad Prism 8.
In silico analysis
The relationship between CTSV mRNA expression and GATA3 protein expression was assessed using cBioPortal (
https://www.cbioportal.org) [
27,
28], via examination of ER-positive breast cancers from the TCGA 2012 breast invasive carcinoma dataset. Two hundred ninety-eight samples within this cohort contained matched CTSV mRNA expression (microarray) and GATA3 protein expression (reverse phase protein array (RPPA)) and were subsequently used for analysis. Linear regression analysis and Spearman’s correlation were performed using GraphPad Prism 8.
Discussion
The work presented here examines for the first time the relationship between CTSV expression and relapse free survival of breast cancer patients. We have shown that CTSV expression has distinctly different outcomes across breast cancer subtypes, particularly in relation to the ER status of the tumour. This would suggest that CTSV may have distinctive roles across breast cancer subtypes; dissecting these roles would require extensive additional research using models representing the different breast cancer subtypes. Given that elevated CTSV expression was associated with reduced survival in ER-positive tumours, we wanted to determine if CTSV may represent a future therapeutic target in this subtype of the disease. Therefore, the research presented herein has focused on dissecting the mechanism of CTSV in ER-positive breast cancer, utilising cell line models representative of the luminal A subtype due to the greater impact of CTSV expression in relation to patient RFS.
Endocrine therapies developed for ER-positive breast cancers such as Tamoxifen, Fulvestrant and aromatase inhibitors such as Anastrozole and Exemestane have exhibited significant clinical success [
36]. However, 30–50% patients treated with these endocrine therapies subsequently develop resistance and disease recurrence can occur up to 20 years post-diagnosis, with recurrence rates of 10–41% dependent on tumour stage at diagnosis and nodal involvement [
37]. Whilst only approximately 10% of ER-positive tumours are metastatic at diagnosis, 20–40% of patients will ultimately develop recurrence at distant organs and such metastatic tumours are classified as incurable [
36]. Therefore, new treatment strategies are urgently required to help patients that exhibit disease recurrence and metastasis.
One of the key findings from this work is the impact of CTSV on the expression of luminal biomarker, GATA3. GATA3 protein expression is a favourable prognostic indicator in ER-positive breast tumours, and numerous studies have reported that loss of GATA3 expression is associated with poor prognosis and propensity for tumour metastasis [
18,
20,
23]. Research has revealed that GATA3 can regulate a number of genes associated with breast to lung metastasis [
23], as well as impede breast cancer metastasis through the inhibition of EMT [
22]. Whether CTSV can facilitate metastasis via suppression of GATA3 remains to be determined through the interrogation of in vivo model systems. Whilst previous studies have associated CTSV expression with distant metastasis [
9] and postulated that CTSV is a liver-tropic gene in lung cancer [
38], no studies have been undertaken as yet to determine the contribution of CTSV to the metastatic process. However, our observation that CTSV depleted cells also exhibit reduced eATP, which has been shown to contribute to metastasis and pre-metastatic events such as EMT, migration and invasion [
35,
39], is suggestive that examining the impact of CTSV on metastasis is warranted.
Our results have identified that CTSV mediates GATA3 phosphorylation at Ser308, which facilitates GATA3 ubiquitination and subsequent degradation by the proteasome. Increased GATA3 turnover mediated by increased proteasomal degradation has previously been reported in breast cancer cells and T cells. Initial studies in T cells postulated that the C-terminal region of GATA3, between residues 261 and 315, is critical for proteasomal degradation [
31], and further work in ER-positive breast cancer cells subsequently identified serine residue 308 as the critical site [
29]. Research has suggested that examination of pSer308 GATA3 expression in breast cancer patients could be predictive of GATA3 loss, enabling stratification of GATA3 positive tumours, as those predicted to lose GATA3 expression would be expected to display a poorer prognosis. Therefore, it would be of interest to undertake immunohistochemical analysis, to determine if pSer308 GATA3 and CTSV expression correlates in clinical samples and be predictive of GATA3 loss, which would be associated with a worse prognosis.
The association between CTSV and the Akt-GSK-3β pathway has not previously been reported. However, Akt and GSK-3β have both previously been associated with GATA3, with Akt reported to facilitate GATA3 phosphorylation in T cells [
40] and GSK-3β known to promote GATA3 degradation in ER-positive breast cancer cells. Activation of Akt is usually associated with a pro-tumour phenotype; however, recent work has suggested that elevated pAkt may be associated with a good prognosis in luminal A ER-positive breast cancer, particularly within the context of PIK3CA mutations [
41].
The E3 ligase component FBXW7α has previously been reported to facilitate GATA3 degradation by the proteasome in a GSK-3β-dependent manner, with GSK-3β acknowledged as the predominant kinase in FBXW7α substrate recognition [
42]. Akt phosphorylation of GSK-3β results in its inactivation via formation of an autoinhibitory pseudosubstrate and subsequent degradation by the proteasome [
43,
44]. Therefore, elevated pAkt resulting in depleted GSK-3β expression would lead to reduced activity of FBXW7α, and accumulation of GATA3 expression, as is evident in our shCTSV cell line models. Inhibition of FBXW7α by the pseudophosphatase STYX has previously been illustrated [
33]; in our models, STYX expression in CTSV-harbouring cells stabilised GATA3 protein expression, to levels similar as that observed in CTSV-depleted cells. Comparable effects were noted in both MCF-7 and ZR-75-1 cells, strengthening our hypothesis that FBXW7α is involved in CTSV-mediated GATA3 turnover.
Previous studies have identified that PI3K inhibition can reduce GATA3 protein expression in T cells and prostate carcinomas [
34,
45]. Our observation that PI3K inhibition can attenuate the elevated GATA3 protein expression associated with shCTSV cells complements previous studies and suggests that the stabilisation of GATA3 protein expression in CTSV-depleted cells is manifested through PI3K-Akt activity. However, what remains to be determined is how CTSV depletion leads to further activation of the PI3K-Akt-GSK-3β pathway, resulting in GATA3 stabilisation. What is of particular interest is that the sustained oncogenic activation of Akt in the two cell lines examined here arise via different mechanisms. MCF-7 cells possess a hyperactivating PI3KCA mutation, whereas ZR-75-1 cells have a loss of PTEN [
46]. Given that we have observed similar effects in both of these cell line models with PI3K inhibition is suggestive that CTSV functions upstream of PI3K-Akt to manipulate GATA3 protein expression. Due to the lack of PTEN in ZR-75-1 cells, it is unlikely that CTSV is mediating an effect via manipulation of PTEN, but it could have an impact on other negative regulators of the PI3K-Akt pathway such as SHIP2 or INPP4B. Furthermore, it is also plausible these effects could be mediated via the action of extracellular CTSV on receptor tyrosine kinases or G-protein coupled receptors, given that these receptors are the most common upstream activator of intracellular signalling pathways like PI3K-Akt. Evidence has shown that cysteine cathepsins including CTSV can be secreted from tumour cells [
47], and recent publications have identified ectodomain shedding is undertaken by cathepsin family members to influence intracellular signalling pathways [
48,
49]. Whilst our emphasis has been on the Akt-GSK-3β pathway, it is also possible that other kinases may facilitate the phosphorylation of GATA3, given the signalling intricacies of tumour cells, with PKA and p38 MAPK both previously identified as facilitating GATA3 phosphorylation [
29,
50].
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