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ORIGINAL ARTICLE
Ahead of print publication  

The regulatory effect of cabazitaxel on epithelial-mesenchymal transition in metastatic prostate cancer


1 Department of Medical Biology, Faculty of Medicine, Bursa Uludag University, Bursa, Turkey
2 Department of Medical Biology, Faculty of Medicine, Sakarya University, Adapazarı, Turkey

Date of Submission03-Mar-2021
Date of Acceptance17-Mar-2021
Date of Web Publication06-Dec-2021

Correspondence Address:
Unal Egeli,
Department of Medical Biology, Faculty of Medicine, Bursa Uludag University, Gorukle, Bursa
Turkey
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_364_21

 > Abstract 


Introduction: Epithelial-mesenchymal transition (EMT) is a critical mechanism that promotes cancer cells to metastasis. Therefore, EMT regulation has become an important target in anticancer therapy approaches in recent years. However, in metastatic prostate cancer (PC), the EMT regulatory effect has not fully understood for cabazitaxel (Cbx), a third line taxane-based chemotherapeutic for metastatic castration-resistant PC.
Aim: In this study, we investigated the antimetastatic and EMT-regulatory effects of Cbx on hormone-sensitive metastatic PC cells.
Materials and Methods: The anticancer effects of Cbx were assessed by WST-1 and Annexin V analysis. The antimetastatic effect of Cbx was evaluated by wound healing and quantitative reverse transcription polymerase chain reaction through EMT-mesenchymal-to-epithelial transition (MET) markers as well as EMT-repressor microRNAs (miRNAs) in Cbx-treated LNCaP cells.
Results: Our results showed that, in addition to its apoptotic and anti-migratory activities, Cbx exhibited the EMT-repressor effects through the prominent downregulation of matrix metalloproteinase-9 and Snail levels as EMT-promoting factors, and the significant upregulation of the certain miRNAs, including miR-205, miR-524, and miR-124, which play a role in EMT-repressing by targeting regulators of the EMT-associated genes.
Conclusion: Although further evaluations are needed to improve the findings, we showed that, in addition to its classical taxane function, Cbx has a regulatory effect on EMT-MET cycling in hormone-sensitive metastatic PC.

Keywords: Cabazitaxel, epithelial-mesenchymal transition, microRNA, prostate cancer, snail



How to cite this URL:
Eryilmaz IE, Eskiler GG, Egeli U, Cecener G. The regulatory effect of cabazitaxel on epithelial-mesenchymal transition in metastatic prostate cancer. J Can Res Ther [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=331857




 > Introduction Top


Prostate cancer (PC) is the most common male malignancy. Despite the improvements in diagnosis and treatment, PC is still a significant cause of morbidity and mortality worldwide.[1] The initial treatment for metastatic PC is androgen deprivation therapy (ADT). However, many patients ultimately develop castration-resistant prostate cancer (CRPC) during ADT.[2],[3] Microtubule-stabilizing taxane-based chemotherapeutics are used as a first-line option for CRPC. Cabazitaxel (Cbx), the second-generation taxane providing additional survival benefits, is clinically used only for CRPC patients who failed to respond to first-generation taxane (Docetaxel), and/or second-generation anti-androgens.[4] However, recent studies have shown that the efficiency of Cbx in PC cells is not limited to the classical taxane functions, including mitotic arrest and apoptosis.[5],[6],[7]

Epithelial-mesenchymal transition (EMT) is a critical process in cancer dynamics involved in acquiring invasive and migratory properties of the epithelial-derived cancer cells. EMT enables the separation of cancer cells from their native microenvironment without an epithelial cell layer requirement. Thus, reprogrammed cells gain mesenchymal and metastatic features with this phenotypic switch.[8],[9] In PC, EMT is correlated with a higher Gleason score, biochemical recurrence, and metastasis that contribute to CRPC progression.[10],[11] Transforming growth factor-β (TGF-β), the common extracellular inducer of EMT, is also an AR cross-talk signaling pathway that leads to accelerating metastatic spread in PC cells.[9],[12],[13],[14] Besides, SMAD-mediated TGF-β signaling activation directly enables the upregulation of EMT transcriptional regulators (Zeb-1/2, Snail, Slug, Twist) resulted in the loss of epithelial markers (E-cadherin, β-catenin) and the acquirement of mesenchymal markers (N-cadherin, Vimentin [VIM]).[15] Thus, primary PC cells acquire more invasive and metastatic features through endogenous TGF-β signaling and EMT promotion.

In the literature, some studies suggest that intermittent ADT or combined therapy with taxane-based chemotherapeutics might offer a better option for advanced PC due to delaying metastasis before CRPC progression.[16],[17],[18] Moreover, Sweeney et al. have reported that combined therapy with Cbx and anti-androgenic agents induced mesenchymal-to-epithelial transition (MET), glandular re-differentiation, and AR nuclear localization in advanced PC, suggesting that new generation taxanes might improve the existing therapy modalities and reorganize algorithms in metastatic PC treatment.[17] Therefore, apart from the classical taxane function, Cbx, as a monotherapy, may have the potential in regulating metastasis-related processes such as EMT in advanced PC.

In the present study, we investigated the EMT regulatory effect of in vitro Cbx monotherapy in LNCaP metastatic PC cells. We focused on the migration ability, the expressions of EMT-MET markers at the RNA level, and EMT-repressor microRNAs (miRNAs), which regulate TGF-β signaling [Supplementary Figure 1] and downstream targets of EMT in Cbx-treated PC cells. Our results showed that Cbx might have an anti-migratory effect by downregulating EMT-inducing transcription factor Snail and matrix metalloproteinase (MMP)-9, and upregulating EMT-repressor miRNAs in metastatic PC.




 > Materials And Methods Top


Drug

Cbx (S3022, 10 mg) was purchased from Selleck Chemicals (Houston, TX, USA). For in vitro treatment, it was prepared in the required amount of dimethyl-sulphoxide (DMSO) to obtain a 5 mM stock solution as noted in the manufacturer's instruction and stored at − 80°C. Before each experiment, a 100 nM intermediate stock solution of Cbx was freshly diluted with the growth medium and applied directly to the cells as 1, 5, and 10 nM on cell culture plates. In all treatments, the rate of DMSO applied to the cells did not exceed 0.01% (v/v).

Cell culture

Human metastatic PC cell line, LNCaP (CRL-1740™), and human prostate epithelial normal cell line, RWPE-1 (CRL-11609™) were purchased from the American Type Culture Collection. LNCaP was cultured in Roswell Park Memorial Institute-1640 medium containing sodium pyruvate and L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), and 1% antibiotic-antimycotic solution (Wisent Bio Product, Quebec, Canada). For the RPWE-1 culture, a keratinocyte serum-free medium kit (Thermo Fisher Scientific, Waltham, MA, USA), which is supplemented with epidermal growth factor and the bovine pituitary extract, was used. The cells were incubated at 37°C and 5% CO2 in a humid incubator (Panasonic, Osaka, Japan). The growth medium was changed every 2–4 days, and cells were passaged using a Trypsin-ethylenediaminetetraacetic acid 0.25% (Thermo Fisher Scientific, Waltham, MA, USA). The two cell lines were screened for checking mycoplasma contamination using both MycoProbe Mycoplasma Detection Kit (R and D Systems, MN, USA) and Hoechst 33258 (Merck KGaA, Darmstadt, Germany) staining and confirmed as negative [Supplementary Figure 2].



Cell proliferation

To determine the appropriate Cbx concentration providing nearly 50% inhibition of cancer cell viability, we used WST-1 cell proliferation colorimetric reagent (BioVision, San Francisco, CA, USA). Briefly, 2 × 104 LNCaP cells/well were seeded into 96-well plates in a volume of 100 μl growth medium and incubated for 48 h, until the cell gains its normal morphology due to low attachment of LNCaP cells. For RWPE-1 cells, 2 × 104 cells/well were seeded and incubated overnight. Subsequently, the seeded cells were treated with 1, 5, and 10 nM Cbx for 24 h. Then, 10 μl of WST-1 reagent was added to each well and incubated for 2–3 h in a growth atmosphere, and cell viability was measured at 450 nm using a TriStar2 S LB 942 monochromator multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany). All doses were measured in three replicates.

Apoptosis

To determine the total apoptotic cells (%) after 1, 5, and 10 nM Cbx treatments, Muse® Annexin-V and Dead Cell Kit (Merck Millipore, Darmstadt, Germany) was used according to the manufacturer's instruction. The cells were seeded into 6-well plates to obtain maximum viability from each nontreated control. After the drug administration, the cells were stained with Annexin V for 30 min in the dark and analyzed in three replicates using a Muse® Cell Analyzer (Merck Millipore, Darmstadt, Germany).

Wound healing assay

Briefly, 1 × 106 cells/well were seeded on 6-well plates and cultured until the cells reach at least 80% density. A single scratch was gently applied to the surface of each well with a yellow pipette tip, and the cells were treated individually with the mentioned Cbx doses in a medium without or with FBS. After 24 h, the migration of the cells to the scratch and the changes in wound area were visualized under an inverted microscope (Nikon Eclipse, Tokyo, Japan), and the distance at 24 h was measured by using the ImageJ program ImageJ program (U. S. National Institutes of Health, Bethesda, MD, USA). Then, the percentage of wound area was calculated for both treatment groups containing FBS or not. The proportional calculation was performed in treatment groups compared to their untreated control accepted as 100%.

Gene expression

To investigate the changes in EMT-related gene expression level after Cbx treatment, total RNA was isolated from Cbx-treated LNCaP cells by using E. Z. N. A.® Total RNA Kit I (Omega Bio-Tek Inc., Norcross, GA, USA). All RNAs were checked for quality and quantity using a spectrophotometer (Beckman Coulter, Inc., Fullerton, CA, USA). For each group, approximately 1 μg RNA was used for cDNA synthesis with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Then, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed by using TaqMan™ Gene Expression Assays, CDH1 (E-Cadherin, Hs01023895_m1), SNAI1 (Snail, Hs00195591_m1), (VIM, Hs00958111_m1) and (MMP-9, Hs00957562_m1) in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Gene expression levels were normalized to (Actin beta, Hs01060665_g1) expression.

MicroRNA expression

To evaluate the effect of Cbx on EMT-related miRNA expression levels, the cells were seeded into 6-well plates at a density of 1 × 106 cells/well. After treatment, small RNAs including miRNAs were isolated from the treated cells by using a NucleoSpin® miRNA Small RNA isolation kit (Macherey-Nagel, Düren, Germany) and instantly reverse transcribed with a miScript® II RT Kit (Qiagen, Hilden, Germany). Then, qRT-PCR was performed for hsa-miR-205-3p (MS00016793), hsa-miR-524-5p (MS00031969), hsa-miR-124-3p (MS00006622), hsa-let-7a-5p (MS00031220), hsa-miR-340-5p (MS00031759), hsa-miR-579-3p (MS00007805), hsa-miR-429 (MS00004193) and Hs_RNU6-2_11 (MS00033740) as an internal control miRNA by using miScript Primer Assays (Qiagen, Hilden, Germany). All expression analysis was performed in triplicate according to miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) protocol on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Statistics

For statistics, GraphPad Prism 6 (La Jolla, CA, USA) was used in all experiments. The Cbx-treated groups were compared with the nontreated control group using an analysis of variance with an appropriate post hoc test. All analyzed values were expressed as the mean ± standard deviation. The statistics of gene and miRNA expressions were carried out in a web-based tool at https://dataanalysis2.qiagen.com/pcr, which uses the 2–ΔΔCt method to compare the gene expression differences between treated groups and nontreated control.


 > Results Top


Cytotoxic and apoptotic effects of cabazitaxel

Cbx significantly inhibited LNCaP viability dose-dependent manner, as shown in [Figure 1]a. 10 nM Cbx treatment decreased the cell viability to 45.5% ± 0.74% for 24 h (P < 0.01). However, the viability of RWPE-1 cells decreased to 69.8% ± 2.5% (P < 0.01). Therefore, Cbx was found to have a more cytotoxic effect on cancer cells in all treatment groups compared to normal cells. Morphological changes in LNCaP cells were also observed after Cbx treatment in [Figure 1]b. Furthermore, Cbx induces apoptotic cell death in both cell lines, as expected. As shown in [Figure 1]c, 5 and 10 nM Cbx significantly triggered apoptosis from 2.38 ± 0.35 to 39.8 ± 1.96 and 43.06 ± 1.04 in LNCaP, respectively (P < 0.01). In RWPE-1 cells, the percentage of total apoptotic cells was 30.8 ± 2.2 after 10 nM Cbx treatment for 24 h as shown in [Figure 1]d (P < 0.01). Thus, Cbx exhibited a higher apoptotic cell death in PC cells for 24 h.
Figure 1: Cytotoxic and apoptotic effects of cabazitaxel on LNCaP and RWPE-1 cells for 24 h. (a) Cell viability results of Cabazitaxel treatment in dose-dependent manner for LNCaP and RWPE-1. (b) Altered morphology of LNCaP cells after cabazitaxel treatment for 24 h. Apoptotic effect of cabazitaxel on (c) LNCaP and (d) RWPE-1 cells, respectively. *P < 0.05, **P < 0.01

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Anti-migratory effect of cabazitaxel

Based on the wound healing assay results, Cbx treatment significantly inhibited the migration ability of hormone-dependent LNCaP cells in a dose-dependent manner in both treatment conditions with and without FBS [Figure 2]a and [Figure 2]b. However, as shown in [Figure 2]a, in the FBS-free group, the proliferation of the untreated control was less, and the migration capacity of the cells, which is not dependent on cell growth, was more pronounced. Thus, the anti-migratory effect of Cbx was observed better in the FBS-free treatment group [Figure 2]a and [Figure 2]b. In these group, the wound area was increased from 100% to 136.32 ± 1.64 (P < 0.01), 161.6 ± 3.01 (P < 0.01), 182.24 ± 2.41 (P < 0.001) at 1, 5 and 10 nM Cbx treatment, respectively for 24 h [Figure 2]c and [Figure 2]d. Simultaneously, the expression level of MMP-9, a common invasion marker of PC, was significantly downregulated after Cbx treatment in all treatment groups [Figure 2]e (P < 0.01). The expression results were consistent with the results of the wound healing assay.
Figure 2: The anti-migratory effect of cabazitaxel on LNCaP cells visualized under an inverted microscope for 24 h. The wound healing assay results of cabazitaxel-treated LNCaP cells in a growth medium (a) without and (b) with 10% fetal bovine serum. The percentage of open wound area in cabazitaxel-treated LNCaP cells (c) without and (d) with 10% fetal bovine serum. (e) Altered MMP-9 expression in cabazitaxel-treated LNCaP cells. *P < 0.05, **P < 0.01, ***P < 0.001

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The effect of cabazitaxel on epithelial-mesenchymal transition-mesenchymal-to-epithelial transition cycling gene expression levels

We investigated fold changes in gene expression levels for EMT-MET cycling markers, including E-cadherin, Snail, and VIM, after 24 h Cbx treatment in LNCaP cells. As depicted in [Figure 3], Cbx treatment downregulated Snail expression in a dose-dependent manner (P < 0.05). No significant alteration was detected in E-cadherin expression, except in the 5 nM treatment group. 5 nM Cbx caused a slight decrease in E-cadherin level. However, there was no change in the E-cadherin expression at the most effective Cbx treatment. While VIM expression was significantly upregulated in 1 and 5 nM Cbx treatment groups (P < 0.01), 10 nM Cbx did not significantly alter the VIM expression, as shown in [Figure 3]. Thus, we concluded that Cbx might exhibit an EMT-regulatory effect by consistently altering the levels of an EMT transcription factor, Snail.
Figure 3: The effect of cabazitaxel on the levels of EMT/MET associated gene expression (CDH1, SNAI1 and VIM). *P < 0.05, **P < 0.01

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The effect of cabazitaxel on Epithelial-mesenchymal transition-related microRNAs' expression levels

To determine the potential effect of Cbx on the regulation of miRNAs that are related to the EMT process, we analyzed the expression levels of 7 different miRNAs in Cbx-treated LNCaP cells in [Figure 4]. Based on the results, a considerable upregulation was detected in miR-205 (P < 0.01), miR-524 (P < 0.01), and miR-579 expressions at 5 and 10 nM Cbx treatments. Besides, 10 nM Cbx treatment partially caused an increase in miR-124 level (P < 0.01). No significant fold change was detected in let-7a and miR-340 levels. Finally, the level of miR-429 was downregulated in Cbx-treated cells compared to untreated control.
Figure 4: The effect of cabazitaxel on EMT-related microRNAs' expression levels. *P < 0.05, **P < 0.01

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 > Discussion Top


In the present study, we added new findings to the regulatory effect of a second-generation taxane, Cbx, on the activity of EMT through EMT-related genes and EMT-repressor miRNAs.

Microtubule-stabilizing chemotherapeutic agents, including Cbx, exhibit their activity through G2/M arrest and apoptosis in cancer cells. Although recent reports have indicated that Cbx has many other functions in PC treatment, such as inhibiting androgen receptor (AR) expression, changing AR localization, causing multi-nucleation, and centrosome clustering,[5],[6],[7],[17],[18] the efficacy of Cbx in EMT repression has not been evaluated in metastatic hormone-sensitive PC cells.

First of all, we determined the cytotoxic and apoptotic effects of Cbx on LNCaP PC cells for subsequent experiments. As consistent with the previous finding,[19] Cbx treatment significantly inhibited cell viability and stimulated apoptosis in LNCaP cells in a dose-dependent manner for 24 h. Moreover, its cytotoxic and apoptotic effects at 5 and 10 nM treatments were more prominent in cancer cells than RWPE-1 normal prostate cells.

EMT/MET cycling is a vital dynamic characterized by reprogramming an epithelial-derived cancer cell to survive without attachment to the cell layer, enter the bloodstream, and gain metastatic property. Therefore, targeting EMT is accepted as a critical anticancer strategy to prevent cancer spread at an earlier stage.[8],[9],[10] In a recent study, EMT/MET cycling can dynamically change in response to in vitro and in vivo Cbx treatment for both androgen-sensitive and CRPC tumor models due to MET inducing effect of Cbx.[7] In this context, we only focused on the potential therapeutic effects of Cbx on metastatic hormone-sensitive PC cells. Our results indicated that Cbx treatment significantly inhibited the migration ability of LNCaP cells in a dose-dependent manner, and its anti-migratory effect was also correlated with the decreased expression of MMP-9, as shown in [Figure 2]. To the best of our knowledge, the expressions of MMPs are part of the EMT process, and the overexpression of MMP-9 contributes to PC invasiveness in LNCaP cells.[20],[21] Moreover, the level of MMP-9 is regulated by TGF-β signaling in many cancer types, and the transcriptional regulators of EMT, Slug, and Snail, directly mediate the upregulation of MMP-9 level in oral cancer[22],[23] and head and neck squamous cell carcinoma.[24] Furthermore, MMP-9 knockdown inhibits TGF-β1-induced EMT and cell invasiveness and migration in esophageal squamous cell carcinoma.[25] Based on our findings, Cbx treatment significantly inhibited the migration capacity of LNCaP cells by downregulating MMP-9 expression.

When the EMT/MET cycling gene expressions were analyzed after Cbx treatment, we detected a slight reduction in mRNA level of E-cadherin (CDH1) and a noticeable increase in VIM, especially at 1 and 5 nM Cbx-treated groups [Figure 3]. The study of Martin et al. notes that the mRNA expression of E-cadherin expression was downregulated in both Cbx-treated VCaP and LNCaP PC cells. In this study, Cbx-treated androgen-responsive prostate tumors exhibit a phenotypically re-differentiated glandular prostate epithelium with intact luminal secretions in vivo.[7] With several EMT/MET dynamic cycles induced by Cbx, a fully differentiated epithelium is formed in the prostate. Therefore, individual cells could de-differentiate into mesenchymal-like derivatives resulted in a reduction in the E-cadherin and an increase in the VIM expressions during this process.[7] Our findings could support these in vivo findings. However, the effects of Cbx on the EMT process should be further explained by in vivo experiments at protein levels. In addition, further molecular investigations are required to assess the role of signaling pathways induced by the down-regulation of E-cadherin in PC cells. On the other hand, we found that Snail, a transcriptional regulator of EMT and a mediator of MMP-9 expression, was significantly downregulated in a dose-dependent manner in Cbx-treated LNCaP cells. A significant reduction in Snail expression most probably resulted in a decrease in the MMP-9 expression due to the direct connection between Snail and MMP-9 as mentioned above, which contribute to inhibit the migration ability of the PC cells in response to Cbx treatment. In a previous study, another transcription factor of EMT, Twist, has been also found to be downregulated after in vitro Cbx treatment.[7] Thus, we concluded that Cbx could exhibit the EMT-inhibitory effects through the down-regulation of EMT-related transcription factors, including Snail.

Cbx also induced significant fold changes in the expression levels of EMT-repressor miRNAs. We selected 7 different miRNAs that contribute to EMT directly or indirectly by targeting TGF-β signaling, transcriptional regulators of EMT, Zeb1/2, Slug and Snail, and the other EMT-related genes such as high-mobility group nucleosome-binding domain (HMGN5). Cbx treatment increased the expression of miR-205, miR-524, and miR-579 [Figure 4], which mainly repress the transcription factors of the EMT process. Among the Cbx-induced upregulated miRNAs, the two (miR-205 ad miR-524) tumor-suppressive miRNAs for PC are the direct repressors of Zeb1/2 and ZEB2/SMAD4, respectively.[26],[27],[28],[29] Moreover, miR-124 directly targets TGF-mediated Slug, an EMT activator in PC cells,[30] which upregulated in 10 nM Cbx-treated PC cells in our study. On the other hand, the downregulation of miR-429 was detected in all Cbx-treated groups. MiR-429 directly targets EMT in colorectal cancer[31] and esophageal squamous cell carcinoma and the over-expression of miR-429 is associated with aggressive features of PC.[32] Therefore, the down-regulation of miR-429 level inhibits cell proliferation by targeting p27Kip1 in human PC cells due to its oncogenic function.[33] In our study, Cbx treatment caused the downregulation of miR-429 expression in LNCaP cells. The obtained findings were supported by the literature.[26],[27],[28],[29],[30],[31],[32],[33]


 > Conclusion Top


Taken together, our results provided new insights into the EMT-inhibitory effect of Cbx on metastatic PC cells. The novelty of our study was that we, for the first time, evaluated the EMT regulator effect of in vitro Cbx monotherapy by analyzing certain EMT-related genes as well as EMT-related miRNAs in metastatic PC. Further studies could support existing results regarding the signaling pathways initiating the EMT process and the underlying mechanisms of Cbx's potential antimetastatic effect.

Financial support and sponsorship

This study was supported by a grant from the Scientific Research Projects Foundation of Bursa Uludag University, Bursa, Turkey (Project No. BUAP[T]-2015/4).

Conflicts of interest

There are no conflicts of interest.



 
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