Blocking Androgen Receptor/Androgen Receptor Variant7 Decreases Metastasis of Estrogen Receptor Positive Breast Cancer Cells Through Modulating Epithelial to Mesenchymal Transition

doi:10.21203/rs.3.rs-1420312/v1

Download PDF

Research Article

Blocking Androgen Receptor/Androgen Receptor Variant7 Decreases Metastasis of Estrogen Receptor Positive Breast Cancer Cells Through Modulating Epithelial to Mesenchymal Transition

https://doi.org/10.21203/rs.3.rs-1420312/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose: Androgen receptor (AR) is often expressed in breast cancer, but its role in estrogen receptor positive (ER+) type is controversial. Although AR and its splicing variant 7 (ARV7) play a role in the pathobiology of breast cancer, the precise mechanisms are not fully understood. Therefore, the aim of the current study is to determine the influence of the blockers of AR (Enzalutamide) and ARV7 (EPI-001) on metastasis and epithelial to mesenchymal transition (EMT) in T47D, an ER+ breast cancer cell line.

Methods: T47D cells were cultured/treated with Enzalutamide or EPI-001; cytotoxicity and cell cycle analysis were determined using Sulphorhodamine-B (SRB) assay and Flowcytometry analysis, respectively, whereas metastasis was assessed by the Scratch wound healing assay. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of AR, ARV7, C-myc, N-Cadherin, E-Cadherin, NF-κB, ROCK1 and 2 and Western blot was used for evaluating the protein expression of cyclin dependent kinases (CDK4, CDK6, Cyclin E), Fibronectin, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP2 and MMP9).

Results: Our results indicated that treatment with Enzalutamide or EPI-001 didn't significantly affect cell proliferation of T47D. However, cells were arrested at S-phase accompanied by a decline in CDK4, CDK6, Cyclin E and metastasis. Fibronectin, VEGF, MMP2, MMP9, ROCK1 and ROCK2 proteins were downregulated. C-myc and NF-κB levels were declined by EPI-001, but not Enzalutamide.

Conclusion: Blocking AR/ARV7 has no effect on cell proliferation, but decreases metastasis by regulating key markers and processes involved in EMT in ER+ breast cancer cells.

Androgen receptor

Androgen receptor variant 7

metastasis

Epithelial to mesenchymal transition

breast cancer

Androgen receptor (AR) is evolving as a vital element in the pathogenesis of breast cancer, the most common malignancy among women worldwide (Aristomenis et al. 2020). AR is expressed in more than 60% of breast cancers and up to 90% of ER + tumors, signifying a possible androgen responsiveness [Li et al. 2016]. The signaling outcome of AR differs across breast cancer subtypes and of particular importance is its interplay with estrogen receptor signaling (KeeMing et al. 2015). The role of AR in ER + breast cancer is somehow controversial and restraining application of AR directed therapies. A recent research has reported that AR performs a tumor suppressor role in ER + breast cancer (Theresa et al. 2021). AR can bind to a group of estrogen response elements, thereby preventing the activation of target genes that facilitate the stimulatory effects of 17-beta-estradiol on breast cancer cells (Amelia et al. 2009). However, there is also indications suggesting that AR may be oncogenic and may contribute to acquired resistance against ER + treatments for breast cancer (De Amicis et al. 2010; Hu et al. 2011). Additionally, it was demonstrated that blocking of AR stopped the estradiol-induced proliferation of an ER+/AR + cell line model (Cochrane et al. 2014) and suppressed cell proliferation of `MCF7 (Shuyuan et al. 2003). Also, constitutively active AR sustained the metastasis of endocrine-resistant hormone receptor positive breast cancer (Bahnassy et al. 2020). Consequently, understanding different AR signaling effects can help to harness the clinical availability of new potent AR-antagonists to hopefully improve current endocrine therapies for ER + breast cancers.

The AR variants arise owing to alternative splicing and/or structural rearrangements of the AR gene; the variants have variable structures, but each lacks all or a part of the ligand binding domain (LBD) (Dehm et al. 2011). This may produce constitutively active, ligand independent, transcription factors or variants resistant to drugs that reduce androgen production and biosynthesis (Li et al. 2013). Androgen receptor variant 7 (ARV7) is one of the most noticeable splice variants in prompting AR mediated gene transcription even under conditions of androgen deprivation and in driving cancer progression in prostate cancer (Bin et al. 2020). Moreover, their role in breast cancer has been recognized and is considered an area of active research (Dong et al. 2014, Theresa et al. 2015).

Several studies have pointed that the aberrant expression of AR/ARV7 is a chief factor that contributes to progression in many cancers, including breast (Macedo 2006), prostate (Adam et al., 2019), hepatocellular (Yeh and Chen 2010), bladder (Wagih and Kamel 2020), oral squamous cell carcinoma (Wu et al. 2015) and gastric cancer (Tian et al. 2013) by endorsing tumor cell growth. However, few studies have focused on metastasis, which is a complex procedure comprising remodeling of the extracellular matrix and leading to epithelial to mesenchymal transition (EMT). This transition is implicated in carcinogenesis and provides metastatic properties to cancer cells by enhancing mobility, invasion, and resistance to apoptosis (Antonella et al. 2017). Furthermore, EMT derived tumor cells attain stem cell characteristics and display a noticeable therapeutic resistance (Vivek 2018). Several biomarkers and pathways play a role in EMT and accordingly affect metastatic potential of cancer cells (Jing et al. 2020).

Therefore, the aim of this study was to determine the effect of blocking AR and ARV7 by Enzalutamide and EPI-001respectively on EMT and metastasis in T47D, an ER + breast cancer cell line. Thereby, several biomarkers were chosen to cover key processes occurring during EMT.

Cell culture

T47D cells were obtained from the American Type Culture Collection (ATCC, Minnesota USA) and were maintained and routinely checked at the Egyptian National Cancer Institute, Cairo, Egypt. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 1.5 g/l sodium bicarbonate and 1% penicillin/streptomycin and kept in a humidified atmosphere with 5% CO₂ at 37°C.

Drug, chemicals & reagents

Enzalutamide (catalogue #: A3003) and EPI-001 (catalog #: B6041) were purchased from ApexBio (HTX, US). Both inhibitors were dissolved in dimethylsulfoxide (DMSO) to prepare a stock solution of 1 mM. For the cytotoxicity assay, different concentrations were used (6.25, 12.5, 25, 50, 100 and 200 uM). For the other experiments, the two inhibitors were added to the medium at a final concentration of 10 µM and 50 µM for Enzalutamide and EPI-001, respectively based on previous reports (Dawn et al. 2014; Brand et al. 2014).

For the Western blot technique, the primary antibodies obtained from Santa Cruz Biotechnology, (TX, US) were used against AR (catalog #: sc-7305), CDK6 (catalog #: sc-7961), CDK4 (catalog #: sc-23896), cyclin E (catalog #: sc-377100), Fibronectin (catalog #: sc-8422), MMP2 (catalog no: sc-13595), MMP9 (catalog no: sc-393859) and VEGF (catalog #: sc-7269). The primary antibody against ARV7 (catalog #: AG10008) was purchased from A&G Precision Antibody (MD, US).

The human ELISA kits for AR (catalog #: In-Hu4116), ARV7 (catalog #: In-Hu4117), C-myc (catalog #: In-Hu1853), NF-κB (catalog #: In-Hu2637), N-Cadherin (catalog #: In-Hu4118), and E-Cadherin (catalog #: In-Hu1892) were purchased from Innova Biotectech (BJ, China). Additionally, the human ELISA Kits for ROCK1 (Catalog #: OKEH06554) and ROCK2 (catalog #: LS-F22011) were procured from Aviva Systems Biology (LA, US) and LifeSpan BioSciences (WA, US).

Sulphorhodamine-B (SRB) assay

The antitumor activity of Enzalutamide and EPI-001 on T47D cells were evaluated by the SRB assay (Skehan et al. 1990). In brief, cells were seeded for 24 hr in 96-well plate at a density of 3×10³ cells per well. They were treated with different concentrations (6.25, 12.5, 25, 50, 100 and 200 µM) of Enzalutamide or EpI-001. For each concentration two wells were used, then, they were incubated for 48 hr and fixed by 20% trichloroacetic acid and stained with 0.4% SRB dye. Finally, the optical density (O.D.) of each well was measured at 570 nm spectrophotometrically using ELISA microplate reader (TECAN sunrise, Germany). The cell survival fraction was calculated by dividing O.D. of treated cells over that of control cells.

Flowcytometry

Treated and control cells were collected after 24 hr and the cells were then fixed with 70% ice cold ethanol, washed and the pellets were resuspended in trypsin buffer and left for 10 min. After that, trypsin inhibitor and 1% RNase buffer were added and incubated for 10 min. followed by the addition of propidium iodide (100 µg/ml). Samples were then incubated in dark for 30 min. at 4°C and the distribution of each cell cycle phase was then determined using a EPICS® C Flow cytometer (FLA, US).

Scratch wound healing assay

T47D cells were seeded in six wells plate and incubated for 24 hr to allow for adherence. The formed monolayer was then scratched slowly with 1 ml pipette tip through each wheel center in one direction. After that, the wells were washed twice with medium in order to remove any detached cells. The cells were then treated with Enzalutamide or EPI-001 for 24 hr, washed twice with PBS then fixed with 3.7% paraformaldehyde for 30 min. and finally, the cells were stained with 1% crystal violet for 30 min. Photos were taken for the stained monolayer and the reduction in gap area was measured using ImageJ software (MD, US).

Immunocytostaining of ARV7

T47D cells were collected and washed with PBS, resuspended in DMEM and placed on glass slide incubated in H₂O₂ for 15 min. then washed again using PBS to be incubated with monoclonal antibody against ARV7 for an hr and then washed three times using PBS. Biotinylated secondary antibody was added and incubated at room temperature for 10 min. and washed with PBS. After that, 3,3′-Diaminobenzidine (DAB) was dropped and incubated for 5 min. followed by counterstaining the slides with hematoxylin. The expression of ARV7 was displayed by brown nuclear color.

Enzyme-linked immunosorbent assay (ELISA)

Treated and control cells were collected and centrifuged for 5 min. at 10,000 rpm at 4°C. The supernatant was then aspirated and the cell pellet was resuspended in phosphate buffer saline (PBS) and the freeze/thaw process was conducted three times in order to lyse the cells. The freeze/thaw process consisted of freezing the samples at − 70°C then immediately thawing them at 37°C. The samples were centrifuged for 5 min. and the supernatant was collected for ELISA quantification in accordance with the manufacturer’s instructions. Protein levels were determined using Bradford assay and their levels were calculated from the constructed calibration curve using second order polynomial curve-fitting models.

Western blot analysis

Treated and control cell were collected as pellets. The Ready Prep™ protein extraction kit (Catalog #:163–2086, Bio-Rad Inc., CA, US) was used for protein extraction. The quantitative protein analysis was conducted by Bradford protein assay kit (Catalog #: SK3041, Bio basic Inc. ON, Canada). After that, the extracted proteins were separated by SDS-PAGE using TGX Stain-Free™ FastCast™ (Catalog #: 161–0181, Bio-Rad Inc., CA, US) and blotted onto PVDF membranes. The membranes were then blocked with 5% non-fat dry milk and incubated at 4°C with primary antibodies overnight followed by incubation with peroxidase-conjugated secondary antibodies. In addition, β-actin antibody was probed in membrane for normalization. The intensities of the bands of the target proteins were normalized against β-actin and were analyzed on the ChemiDoc MP imager.

Statistical analysis

The data were presented as mean ± S.E.M. Multiple comparisons were performed utilizing one-way analysis of variance (ANOVA) followed by Tukey’s as a post hoc test. F values were expressed to indicate the ratio between and within group variances. Significance was established when p ≤ 0.05. All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 26.0 (NY, US). Graphs were sketched using GraphPad InStat, version 5.0 (LA, US) (Supplemental data).

Effect of EPI-001 and Enzalutamide on the proliferation of T47D

As depicted in Fig. 1 no significant increase in the proliferation of T47D was observed following treatment by EPI-001 or Enzalutamide as measured by SRB assay. For the two blocker no IC50 value was detected.

EPI-001 and Enzalutamide induced S-phase cell cycle arrest and downregulated cell cycle regulatory proteins

Treated T47D groups with EPI-001 and Enzalutamide demonstrated an S-phase cell cycle arrest with 50.38% and 14.01% of cell population retained in S-phase compared to 0.51% in untreated control group (Fig. 2A & 2B). These results were further confirmed by the qualitative evaluation of Cycline E, CDK4 and CDK6 protein expression levels by measuring the optical densities of their Western blot bands (2C). EPI-001 reduced Cycline E, CDK4 and CDK6 to 43.71%, 35.73% and 20.5% of the corresponding control group optical densities, respectively (Fig. 2D). Likewise, Enzalutamide showed a comparable reduction of Cycline E, CDK4 and CDK6 band densities to 38.82%, 33.18% and 19.94 respectively compared to control group (Fig. 2D).

EPI-001 and Enzalutamide inhibited cell migration and motility in T47D

The implication of both inhibitors on cell migration & motility was evaluated using Scratch wound healing assay. The wound area was measured using image j software after 24 hr of inhibitors incubation with T47D cells (Fig. 3A). The scratch area exhibited a reduction of 60.97% in control group after 24 hr. On the other hand, the scratch area reduction percent in EPI-001 and Enzalutamide treated groups were 33.67% and 34.54%, respectively (Fig, 3B); which is significantly different from control group [F(2,17) = 44.358, P ≤ 0.05].

Expression of AR/ARV7 in T47D and the effects of EPI-001 and Enzalutamide on their expression

Immunocytochemistry, Western blot and ELISA techniques confirmed the expression of AR and ARV7 proteins inT47D (Fig. 4A, B). Moreover, the expression levels of AR and ARV7 were not significantly affected following treatment with EPI-001 or Enzalutamide (Fig. 4C) but their effects on cellular functions were further evaluated.

EPI-001 downregulated NF-κB and C-myc protein levels in T47D

The effect of EPI-001 and Enzalutamide on the expression levels of NF-κB and C-myc were measured by ELISA technique and illustrated in Fig. 5. EPI-001 significantly reduced both NF-κB to 50.55% [F(2,5) = 20.58, P ≤ 0.05] and C-myc to 73.88% [F(2,5) = 98.36, P ≤ 0.05] compared to control group while Enzalutamide had no significant effect on their protein expression.

Effect of EPI-001 and Enzalutamide on E-Cadherin and N-Cadherin

The effects of EPI-001 and Enzalutamide on the protein levels of the epithelial marker (E-Cadherin) and mesenchymal marker (N-Cadherin) were elucidated utilizing ELISA technique (Fig. 6). Both EPI-001and Enzalutamide significantly elevated the levels of E-Cadherin by 10.77 and 12.76 folds respectively compared to control group, [F(2,5) = 10155.723, P ≤ 0.05] but no significant effects were observed on N-cadherein levels.

EPI-001 and Enzalutamide downregulated Fibronectin

Alongside the demonstrated effect of EPI-001 and Enzalutamide on E-Cadherin; the Fibronectin protein level was reduced as measured by Western blot by 59.44% and 62.46% for both inhibitors, respectively (Fig. 7).

EPI-001 and Enzalutamide reduced angiogenesis

The possible anti-angiogenic potential of both inhibitors was measured through qualitative measurement of VEGF level (Fig. 8). Indeed, EPI-001 and Enzalutamide markedly reduced VEGF band densities to 51.25% and 42.2% compared to control group, respectively.

EPI-001 and Enzalutamide downregulated protein levels of matrix metalloproteinases (MMPs): MMP2 and MMP9

The quantification of MMP2 and 9 via Western blot analysis in different groups (Fig. 9A) showed a marked reduction of their levels after treatment with both inhibitors. Indeed, both MMPs 2 and 9 were reduced by EPI-001 to 41.16% and 35.79% respectively as compared to the control value. Along side, Enzalutamide reduced the band density to 40.39% and 32.53%, respectively (Fig. 9B).

EPI-001 and Enzalutamide downregulated ROCK1 and ROCK2 levels

The impact of AR and ARV7 on activation of Rho kinase 1and 2 (ROCK1,2) was evaluated through quantification of ROCK1,2 after treatment with EPI-001 and Enzalutamide. As illustrated in Fig. 10; EPI-001 reduced ROCK1 level to 30.18% of control group. Such a reduction was significantly different from either control or Enzalutamide treatment group [F(2,5) = 1181.889, P ≤ 0.05]. Meanwhile, Enzalutamide significantly reduced ROCK1 level to 40.48% of control group. On the other hand, both EPI-001 and Enzalutamide significantly reduced ROCK 2 activity to 23.42% and 28.94% of control value, respectively [F(2,5) = 438.537, P ≤ 0.05]

The expression of AR in breast cancer makes it an attractive therapeutic target. However, its role in breast cancer development is still controversial as it can either inhibit or promote breast tumor growth and metastasis (Pia et al. 2018; Mengyao et al. 2020). Among AR variants, ARV7 is drawing attention as a prospective marker for one of the resistance mechanisms to anti AR therapy of castration resistant prostate cancer (CRPC) (Ye and Tian, 2021) and has also been detected in breast cancer subtypes (Dong et al. 2014). Yet, the role of both AR and ARV7 in ER + breast cancer needs further clarification. In the current investigation, the influence of AR/ARV7 on ER + breast cancer has been studied through blocking AR/ARV7 with Enzalutamide or EPI-001. It was noticed that there was no significant effect in the proliferation of the ER + cell line T47D following treatment by the two blockers. Nevertheless, there was a decrease in the protein expression of CDK4, CDK6 and Cyclin E which are known to play important roles in breast cancer (Khandan et al. 2002; Mara et al. 2019). This was accompanied by a decrease in number of cells in G1 phase and cell cycle arrest in S-phase. In harmony with our results, a previous study indicated that blockade of AR signaling decreased the G1-S cyclins in a resistant breast cancer cell line (Wenfei et al. 2019). This effect of the cell cycle which was not translated into decrease in cell proliferation can be explained by the existence of equilibrium between cell cycle arrest and proliferation governed by several other pathways and growth factors in the tumor microenvironment and extracellular matrix (ECM). Whether the balance is tilted towards cell cycle arrest or cell proliferation is influenced by how the cell incorporates the various signals, internal or external to the CDK network that stimulates or delays progression in the cell cycle (Claude and Albert 2014, 2016).

Breast cancer exhibits metastatic heterogeneity with distinctive precedence to many organs as bone, lung, liver and brain. Only few studies have been performed to evaluate AR expression in primary tumors and metastatic samples. It was demonstrated that AR persisted in most of metastatic samples from AR + triple negative breast cancers (TNBC) (Yasuhiro et al. 2015) as well as breast cancer bone metastasis (Nicola et al. 2018). Moreover, androgens activated myosin, a cytoskeletal protein having a role in cell motility and invasion in T47D cells (Maria et al. 2016). Similar to AR, not many studies have explored the relation between ARV7 and metastasis in breast cancer. However, in prostate cancer, it was revealed that ARV7 positive metastatic CRPC patients may have worse prognosis (Jiatong and Ranlu 2020). These studies and others stimulated our interest to investigate the effect of AR/ARV7 mediated metastasis in ER + breast cancer cell line by blocking AR/ARV7 with Enzalutamide or EPI-001. Our results indicated that these blockers inhibited metastasis in T47D as indicated by Scratch wound healing assay. A previous study reported that Enzalutamide inhibited cell migration and invasion in TNBC cell line in an AR dependent manner (Francesco et al. 2016). Also, targeting ARV7 axis resulted in altering the prostatic cancer cells progression and resistance to androgen deprivation therapy with Enzalutamide (Ronghaoet al. 2017).

The process whereby epithelial cells are transformed into mesenchymal cells (EMT) can impact cancer growth and dissemination whereby epithelial cells change their adhesion profiles from cell/cell contacts to cell/matrix interactions, leading to metastasis and therapy resistance EMT is a dynamic procedure demanding the interaction of main processes and signals in tumorigenesis; angiogenesis, inflammation, immunomodulation, matrix degradation, etc (Erik et al. 2020). In prostate cancer, both AR and AR splice variants contribute to prostate cancer aggressiveness through induction of EMT (Campbell et al. 2013; Dejuan al., 2015). Breast cancer cells also undergo EMT modifications, enhancing tumor progression and metastais (Chiara et al. 2012; Renata et al. 2020). Therefore, In the present investigation, it was of interest to study the effect of AR/ARV7 inhibitors, Enzalutamide and EPI-001, on EMT in T47D to explain their observed antimetastatic effect. To explore this hypothesis, several markers which may impact EMT were studied.

One of the investigated markers is the inflammatory mediaor NF-κB which has a significant role in promoting aggressive phenotypes of ER + breast cancer. Both ER and NF-κB can suppress and activate one another and each of these mechanisms have the potential to contribute to more aggressive ER + breast cancer phenotype and metastasis (Emily et al. 2020). Several reports stated that NF-κB modulates EMT in breast cancer (Bruno et al. 2017; Margit et al. 2004; Li et al. 2012). Consequently, these reports and others support the need to reconsider targeting the NF-κB pathway in ER + breast cancer as an approach to prevent disease progression and relapse. The promoter region of the AR gene possesses NF-κB response elements and stimulation of NF-κB activity was found to elevate AR levels in prostate cancer cells (Liying et al. 2009). Therefore, NF-κB may represent a target for breast cancer therapy through regulation of AR. As displayed by our data, NF-κB protein levels declined following treatment by EPI-001 in T47D while no significant effect was observed by Enzalutamide. Since studies suggest an association between high estrogen receptor expression, invasion, metastasis and NF-κB (Xian-Long et al. 2014), thus the potential effect of EPI-001 as downregulator of NF-κB may be implicated, at least in part, in decreasing metastasis in ER + breast cancer. It's noteworthy that NF-κB controls a number of markers involved in cell cycle progression, inhibition of apoptosis, immunoinflammatory responses and cell adhesion. Some of these markers are cell adhesion molecules, C-myc, VEGF and MMPs (Takashi et al. 2007). Similarly, a pathological increase in cellular motility, leading to malignant transformation, was linked with RhoA–ROCK-mediated activation of NF-κB (Catherine et al. 2009).

E-Cadherin, N-Cadherin and Fibronectin are vital EMT markers. E-Cadherin is an epithelial marker and its decreased level correlates with increased invasiveness and metastasis of tumors. On the other hand, the mesenchymal N-Cadherin is regarded as an oncoprotein where it stimulates invasion, proliferation, angiogenesis and metastasis (Mayra et al. 2019). Similar to N-cadherin, Fibronectin glycoprotein is associated with metastatic tumors and poor prognosis in many cancers including breast cancer (Georgios et al. 2020). Yan-Nian et al. (2008) suggested that activated AR can downregulate E-Cadherin expression and promote activation of EMT and metastasis in breast cancer patients through binding to E-Cadherin regulatory sequences. Additionally, constitutively active AR variants upregulate the expression of N-Cadherin in prostate cancer cells (Félicie et al. 2013). Dibash et al. (2016) identified a novel pathway in prostate cancer whereby miR-1207-3p regulates the AR via a pathway involving Fibronectin. Consequently, in the present study, the effect of AR/ARV7 blockers Enzalutamide and EPI-001 on the expression levels of E-Cadherin, N-Cadherin and Fibronectin was investigated. Both blockers significantly elevated the levels of E-Cadherin but didn't induce significant change in N-Cadherin. Also, the two blockers reduced the levels of Fibronectin by 39.15% and 41.02% respectively.

C-myc is another driver of EMT in mammary epithelial cells (Kyoung et al. 2010) and TNBC (Shuping et al. 2017). It also plays a proliferative role and imparts resistance to chemotherapeutic agents in ER + breast cancer cell lines (Yassi et al. 2017). Therefore, targeting C-myc in combination with inhibitors of other oncogenic pathways may provide a therapeutic strategy for breast cancer. C-myc has a critical role in regulating the coordinated expression of AR and AR variants in CRPC (Shanshan et al. 2019) and AR promotes ligand independent prostate cancer progression through C-myc upregulation (Lina et al. 2013). Likewise, in molecular apocrine breast cancer, the androgen signaling pathway was found to upregulate the activity of myc (Keely et al. 2014). In harmony with our NF-κB data there was a significant downregulation of C-myc protein following treatment with EPI-001 but not Enzalutamide. This indicates that EPI-001 may regulate EMT, partly through inhibition of NF-κB /C-myc pathway.

Our data also indicated a marked decline in the angiogenic marker VEGF following treatment by Enzalutamide and EPI-001. Angiogenic cytokines such as VEGF have been identified in prostatic tumors and the AR is significantly associated with vascular endothelial growth through processes involving SP-1 and HIF-a (Jane et al. 2005; Kurtis et al. 2017). The stimulating effect of androgen on VEGF is significant and there is convincing evidence that part of the antitumor effect of antiandrogen therapy is mediated by its downregulatory effect on VEGF (Woodward et al. 2005). Moreover, it has been suggested that alterations in cellular pathways related to both endocrine and VEGF may contribute to breast cancer progression through EMT (Qian et al. 2013; Minna et al. 2016). Therefore, in the present study, it's possible that the tested blockers modulated EMT by reducing VEGF levels.

MMPs are proteolytic enzymes that degrade the ECM (Masoud et al. 2019) and are implicated in angiogenesis (Saray et al., 2019), invasion and metastasis in various tumors including breast cancer (Duffy et al. 2000). Moreover, elevated levels of MMPs in ECM significantly increase EMT (Cristian et al. 2019). Therefore, MMPs represent targets for cancer therapy (Arthur et al. 2018). It was demonstrated that expression of MMPs is associated with the presence of AR in epithelial ovarian tumors, hepatocellular carcinoma and prostate cancer. The presence of AR in these tumor types was a risk factor for overall survival or predictive of invasion and metastasis. Also, the regulation of MMPs in prostate cancer was abolished by androgen inhibitor Bicalutamide (Flavia et al. 2020; Yan et al. 2015; See-Tong et al. 2004). Moreover, AR has been correlated with the expression of some MMPs and TIMP-2 (Luis et al. 2008). Indeed, in the current study, there was a marked reduction of the levels of MMP2 and MMP9 in T47D cells exhibited by the two blockers Enzalutamide and EPI-001.

The Rho/ROCK are AGC family serine threonine kinases. Previous data indicate that Rho/ROCK signaling, which leads to cytoskeletal rearrangement, plays a central role in breast cancer cells and their disrupted tissue architecture. Therapy targeting the ROCK signaling cascade may provide a therapeutic opportunity for breast cancer (Masahiro and Mina 2016). ROCK pathway has been involved in regulating EMT in renal cell carcinoma by affecting the cytoskeleton during EMT reversal to stabilize the epithelial structure (Shreyas et al. 2009). Also, SHROOM, which is a key mediator of ROCK pathway, was found to be a potent antagonist for EMT in nasopharyngeal carcinoma cells (Jing et al. 2019). Links have been reported between androgens, AR and ROCK pathways: Expression of ROCK1 and ROCK2 are linked to AR expression, androgens induce activation of RhoA and its translocation into the plasma membrane, RhoA acts as a direct AR targeted gene, and Rho signaling has been implicated in functional activation of AR (Wen et al. 2013; Kroiss et al. 2015). It was reported that androgens alter the architecture of cytoskeleton in T47D breast cancer

cell and promote cell migration and invasion through modulation of meosin (Maria et al. 2016). Consequently, in the present work, the effect of AR inhibitors Enzalutamide and EPI-001 on the protein levels of ROCK1 and ROCK2 in T47D cells were investigated. The results showed that both inhibitors reduced the protein expression of the two markers which may be reflected on invasion and metastasis of the malignant cells.

To sum up, our study indicated that blocking AR/ARV7 by Enzalutamide and/or EPI-001 impacted the cell cycle by downregulating cell cycle regulatory genes CDK4, CDK6, Cyclin E and inducing S-phase cell cycle arrest. Most importantly, there was decrease in metastasis by modulating key biomarkers and proteins involved in regulating the ECM and remodeling of EMT. Thus, we suggest that blocking AR/ARV7 may have beneficial antimetastatic effect in ER + breast cancer subtype.

Funding

This work was supported by Cairo University, Grant numbers 170

Competing interests

The authors declare no competing interests.

Author contribution

All authors agreed to the study conception and design and read and approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

BAM: methodology, analysis, investigation, drafting

HSE: design, supervision, review

GM: methodology

MS: methodolgy

SS: design, data analysis, supervision, review

MK; design, supervision, methodology, data analysis, drafting and revision

Ethics approval

Not applicable

Consent to participate

Not applicable.

Competing interests

The authors declare no competing interests

Aristomenis A, Ilianna Z, Athanasios GP, Michalis VK (2020) AR in breast cancer-clinical and preclinical research insights. Molecules 25(2):358. https://doi.org/10.3390/molecules25020358
Li W, O’Shaughnessy J, Hayes D, Campone M, Bondarenko I, Zbarskaya I et al (2016) Biomarker associations with efficacy of abiraterone acetate and exemestane in postmenopausal patients with estrogen receptor-positive metastatic breast cancer. Clin Cancer Res Off J Am Assoc Cancer Res 22:6002–9. https://doi.org/10.1158/1078-0432.CCR-15-2452
KeeMing C, Megan O'B, Myles B, Elgene L (2015) Targeting the AR in breast cancer. Curr Oncol Rep 17(2):4. https://doi.org/10.1007/s11912-014-0427-8
Theresa E Hickey, Luke A Selth, Kee Ming C et al (2021) The AR is a tumor suppressor in estrogen receptor-positive breast cancer. Nat Med 27(2):310-320. https://doi.org/10.1158/0008-5472.CAN-09-0452
Amelia AP, Grant B, Carmela R et al (2009) AR inhibits estrogen receptor-alpha activity and is prognostic in breast cancer. Cancer Res 69(15):6131-40. https://doi.org/10.1158/0008-5472
De Amicis F, Thirugnansampanthan J, Cui Y, Selever J, Beyer A, Parra I et al (2010). AR overexpression induces tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat 121(1):1–11. https://doi.org/10.1007/s10549-009-0436-8
Hu R, Dawood S, Holmes MD, Collins LC, Schnitt SJ, Cole K et al (2011) AR expression and breast cancer survival in postmenopausal women. Clin Cancer Res: Off J Am Assoc Cancer Res 17(7):1867–74. https://doi.org/10.1158/1078-0432.CCR-10-2021
Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D’Amato NC et al (2014) Role of the AR in breast cancer and preclinical analysis of Enzalutamide. Breast Cancer Res BCR16(1):R7. https://doi.org/10.1186/bcr3599
Shuyuan Y, Yueh-Chiang H, Peng-Hui W, Chao X, Qingquan X, Meng-Yin T, Zhihong D, Ruey-Sheng W, Ting-Hein L, Chawnshang C (2003) Abnormal mammary gland development and growth retardation in female mice and MCF7 breast cancer cells lacking AR. J Exp Med 198:1899–1908. https://doi.org/10.1084/jem.20031233
Bahnassy S, Thangavel H, Quttina M, Fatima KA, Dhanyalayam D, Ritho J, Karami S, Ren J, Bawa-Khalfe T (2020) Constitutively active AR supports the metastatic phenotype of endocrine-resistant hormone receptor-positive breast cancer. Cell Commun Signal 18(1):154. https://doi.org/10.1186/s12964-020-00649-z
Dehm SM, Tindall DJ (2011) Alternatively, spliced androgen receptor variants. Endocr Relat Cancer 18:R183–196. https://doi.org/10.1530/ERC-11-014
Li Y, Chan SC, Brand LJ, Hwang TH, Silverstein KA, Dehm SM (2013) AR splice variants mediate Enzalutamide resistance in castration-resistant prostate cancer cell lines. Cancer Res 73:483–489. https://doi.org/10.1016/j.pharmthera.2013.07.003
Bin W, Shiqi W, Yong F, Guangxi S, Dalin H, Jer-Tsong H, Xinyang W, Hao Z, Kaijie W (2020) The AKR1C3/ARV7 complex maintains CRPC tumour growth by repressing B4GALT1 expression J Cell Mol Med 24(20):12032-12043. https://doi.org/10.1111/jcmm.15831
Dong GH, Theresa EH, Connie I, Dhilushi D, Lu L, Wayne DT, Luke AS, Peter IM (2014) Identification of AR splice variant transcripts in breast cancer cell lines and human tissues. Horm Cancer 5(2):61-71. https://doi.org/10.1007/s12672-014-0171-4.
Theresa EH, Connie MI, Heidi D et al (2015) Expression of AR splice variants in clinical breast cancers. Oncotarget. 6(42):44728-44. https://doi.org/10.18632/oncotarget.6296
Macedo LF (2006) Role of androgens on MCF-7 breast cancer cell growth
and on the inhibitory effect of letrozole. Cancer Res 66:7775–7782. https://doi.org/10.1158/0008-5472.CAN-05-3984
Adam S, Ilsa C, Wei Y et al (2019) AR splice variant-7 expression emerges with castration resistance in prostate cancer. J Clin Invest 129(1):192-208. https://doi.org/10.1172/JCI122819
Yeh S, Chen P (2010) Gender disparity of hepatocellular carcinoma: the roles of sex hormones. Oncology Basel 78:172–179. https://doi.org/10.1159/000315247
Wagih M, Kamel M (2020) Evaluation of AR status in urothelial carcinoma of the urinary bladder in Egyptian patients: an immunohistochemical study. African journal of urology 26(1):1. https://doi.org/10.1186/s12301-019-0014-1
Wu T, Luo F, Chang Y et al (2015) The oncogenic role of ARs in promoting the growth of oral squamous cell carcinoma cells. Oral Dis 21:320–327. doi: 10.1111/odi.12272
Tian Y, Wan H, Lin Y et al (2013) AR may be responsible for gender disparity in gastric cancer. Med Hypotheses 80:672–674. https://doi.org/10.1111/odi.12272
Antonella S, Francesca D, Gwenola M, Paola N (2017) Deciphering the loop of epithelial-mesenchymal transition, inflammatory cytokines and cancer immunoediting. Cytokine Growth Factor Rev 36:67-77. https://doi.org/10.1016/j.cytogfr.2017.05.008
Vivek M (2018) Epithelial mesenchymal transition in tumor metastasis. Annu Rev Pathol. 13:395-412. https://doi.org/10.1146/annurev-pathol-020117-043854
Jing Y, Parker A, Geert Berx et al (2020) Guidelines and definitions for research on epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 21(6):341-352. https://doi.org/10.1038/s41580-020-0237-9
Dawn RC, Sebastián B, Britta MJ et al (2014) Role of the AR in breast cancer and preclinical analysis of Enzalutamide. Breast Cancer Research 16: R7. https://doi.org/10.1186/bcr3599
Brand LJ, Olson ME, Ravindranathan P, Guo H, Kempema AM et al (2015) EPI-001 is a selective peroxisome proliferator-activated receptor-gamma modulator with inhibitory effects on androgen receptor expression and activity in prostate cancer. Oncotarget 6: 3811-3824. https://doi.org/.10.18632/oncotarget.2924
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D et al (1990) New colorimetric cytotoxicity assay for anticancer drug screening. J Natl Cancer Inst 82:1107–12. https://doi.org/10.1093/jnci/82.13.1107
Pia G, Marzia DD, Giovanni G, Erika Di Z, Antonio B, Antimo M (2018) The AR in breast cancer. Front Endocrinol (Lausanne) 9:492 https://doi.org/10.3389/fendo.2018.00492
Mengyao C, Yunben Y, Kai X, Lili L, Jian H, Fuming Q (2020) AR in breast cancer: from bench to bedside. Front Endocrinol (Lausanne). 11:573 https://doi.org/10.3389/fendo.2020.00573
Ye C, Tian L (2021) Molecular origin, expression regulation, and biological function of AR splicing variant 7 in prostate cancer. Urol Int 105(5-6):337-353. https://doi.org/10.1159/000510124
Dong GH, Theresa EH, Connie I, Dhilushi DW, Lu L, Wayne DT, Luke AS, Peter IM (2014) Identification of AR splice variant transcripts in breast cancer cell lines and human tissues. Horm Cancer 5(2):61-71. https://doi.org/10.1007/s12672-014-0171-4
Khandan K, Susan LT, Thomas AB et al (2002) Cyclin E and survival in patients with breast cancer. N Engl J Med 347(20):1566-75. https://doi.org/10.1056/NEJMoa021153
Mara B, Silvia LM, Claudia F, Roberta A (2019) Multiple effects of CDK4/6 inhibition in cancer: From cell cycle arrest to immunomodulation. Biochem Pharmacol 170:113676. https://doi.org/10.1016/j.bcp.2019.113676
Wenfei J, Yaqin S, Xin W, Weiwei H, Lin T, Shengwang T, Hua J, Yongqian S, Xiaoxiang G (2019) Combined AR blockade overcomes the resistance of breast cancer cells to palbociclib. Int J Biol Sci 15(3):522-532. https://doi.org/10.7150/ijbs.30572
Claude G, Albert G (2014) The balance between cell cycle arrest and cell proliferation: control by the extracellular matrix and by contact inhibition. Interface Focus 4(3):20130075. https://doi.org/10.1098/rsfs.2013.0075
Claude G, Albert G (2016) Dynamics of the mammalian cell cycle in physiological and pathological conditions. Wiley Interdiscip Rev Syst Biol Med 8(2):140-56. https://doi.org/10.1002/wsbm.1325
Yasuhiro M, Yasuhiro N, Takashi S, Noriko N et al (2015) AR and enzymes in lymph node metastasis and cancer reoccurrence in triple-negative breast cancer. Int J Biol Markers 30(2):e184-9. https://doi.org/10.5301/jbm.5000132
Nicola A, Aditya B, Ben SW et al (2018) AR expression in breast cancer CTCs associates with bone metastases. Mol Cancer Res 16(4):720-727. https://doi.org/10.1158/1541-7786.MCR-17-0480
Maria MM, Jorge E, Maria SG, Andrea G, Paolo M, Eleonora R, Alessandro DG, Tommaso S (2016) Androgens regulate T47D cells motility and invasion through actin cytoskeleton remodeling. Front Endocrinol 7:136. https://doi.org/10.3389/fendo.2016.00136
Jiatong Z, Ranlu L (2020) The association between AR splice variant 7 status and prognosis of metastatic castration-resistant prostate cancer: A systematic review and meta-analysis. Andrologia 52(7):e13642. https://doi.org/10.1111/and.13642
Francesco C, Alyson M, Stephen FM, Naoise CS, Elizabeth JR, Norma O, John C, Michael JD (2016) Preclinical evaluation of the AR inhibitor Enzalutamide in triple-negative breast cancer cells. Endocr Relat Cancer 23(4):323-34. https://doi.org/10.1530/ERC-16-0068
Ronghao W, Yin S, Lei L, Yuanjie N, Wanying L, Changyi L, Emmanuel SA, Jun L, Shuyuan Y, Chawnshang C (2017) Preclinical study using malat small interfering RNA or AR splicing variant degradation enhancer ASC-J9 to suppress Enzalutamide-resistant prostate cancer progression. Eur Urol 72(5):835-844. https://doi.org/10.1016/j.eururo.2017.04.005
Erik H, Rajender N, Süleyman E (2020) Extracellular matrix in the tumor microenvironment and Its impact on cancer therapy. Front Mol Biosci 6:160. https://doi.org/10.3389/fmolb.2019.00160
Campbell MG, Natasha K (2013) Epithelial mesenchymal transition (EMT) in prostate growth and tumor progression. Transl Androl Urol 2(3):202-211 https://doi.org/10.3978/j.issn.2223-4683.2013.09.04
Dejuan K, Seema S, Yiwei L, Wei C, Wael AS, Elisabeth H, Fazlul HS (2015) AR splice variants contribute to prostate cancer aggressiveness through induction of EMT and expression of stem cell marker genes. Prostate 75(2):161-74. https://doi.org/10.1002/pros.22901
Chiara F, Massimo B, Daniele G, Giovanna D (2012) Epithelial-mesenchymal transition and breast cancer: role, molecular mechanisms and clinical impact. Cancer Treat Rev 38(6):689-97. https://doi.org/10.1016/j.ctrv.2011.11.001
Renata MB, Edward H, Andreza MV, Pedro B, Jose M, Christina B (2020) Extracellular matrix derived from high metastatic human breast cancer triggers epithelial-mesenchymal transition in epithelial breast cancer cells through αvβ3 integrin. Int J Mol Sci 21(8):2995. https://doi.org/10.3390/ijms21082995
Emily S, Svetlana ES, Jonna F (2020) Update on the Role of NFκB in Promoting aggressive phenotypes of estrogen receptor-positive breast cancer. Endocrinology. 161(10):bqaa152. https://doi.org/10.1210/endocr/bqaa152
Bruno RB, Andre LM, Gerson MF, Waldemir F, José AM, Amanda MM, Stephany C, Eliana SF (2017) NF-kappaB is involved in the regulation of EMT genes in breast cancer cells. PLoS One. 12(1): e0169622. https://doi.org/10.1371/journal.pone.0169622
Margit AH, Ninel A, Bernd B, Stefan G, Andreas S, Hubert P, Norbert K, Hartmut B, Thomas W (2004) NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest 114(4):569-81. https://doi.org/10.1172/JCI21358
Li C, Xia W, Huo L et al (2012) Epithelial-mesenchymal transition induced by TNF-α requires NF-κB-mediated transcriptional upregulation of Twist1. Cancer Res 72(5): 1290-1300. https://doi.org/10.1158/0008-5472.CAN-11-3123
Liying Z, Saleh A, Fangming D, Lishi C, Priti L, Umeshkumar KB, Ruslan K, Sven W, Hans GL, Chawnshang C, Howard IS, William LG (2009) NF-kappaB regulates AR expression and prostate cancer growth. Am J Pathol 175(2):489-99. https://doi.org/10.2353/ajpath.2009.080727
Xian-Long Z, Wei F, Gui Y, Ming-Xia Y (2014) The clinical significance of PR, ER, NF-κB and TNF-α in breast cancer. Dis Markers 2014:494581. https://doi.org/10.1155/2014/494581
Takashi O, Takaomi S, Kaori A (2007) NF-kappa B signaling and carcinogenesis. Curr Pharm Des 13(5):447-62. https://doi.org/10.2174/138161207780162944
Catherine FC, Irene KY, Tim E, Amanda C, Heike D, Peter S (2009) Loss of cell-cell contacts induces NF-kappaB via RhoA-mediated activation of protein kinase D. J Cell Biochem 106(4):714-28. https://doi.org/10.1002/jcb.22067
Mayra P, Sergio S (2019) Extracellular matrix alterations in metastatic processes. Int J Mol Sci 20(19):4947. https://doi.org/10.3390/ijms20194947
Georgios E, Angélique S, Michaël R, Zeinab R, Delphine C, Ellen V (2020) Shaping up the tumor microenvironment with cellular fibronectin. Front Oncol 10:641. https://doi.org/10.3389/fonc.2020.00641
Yan-Nian L, Ying L, Han-Jung L, Yung-H, Ji-Hshiung C (2008) Activated AR downregulates E-Cadherin gene expression and promotes tumor metastasis. Mol Cell Biol 28(23):7096-108. https://doi.org/10.1128/MCB.00449-08
Félicie C, Irène A, Eva E, Jean-Pierre B, Jean-Emmanuel K, Jocelyn C (2013) Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in prostate cancer cells. PLoS One 8(5):e63466. https://doi.org/10.1371/journal.pone.0063466
Dibash K, Michelle N, Adeodat I, Jong Y, Thahmina A, Konstantinos K, Brian DR, Joseph RO, Olorunseun OO (2016) miR-1207-3p regulates the AR in prostate cancer via FNDC1/fibronectin. Exp Cell Res 348(2):190-200. https://doi.org/10.1016/j.yexcr.2016.09.021
Kyoung B, Min K, Won Y, Keon W (2010) Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells. Cancer Lett 293(2):230-9. https://doi.org/10.1016/j
Shuping Y, Vino TC, Liping X, Arun KR, Kaladhar BR (2017) Myc mediates cancer stem-like cells and EMT changes in triple negative breast cancers cells. PLoS One 12(8):e0183578. https://doi.org/10.1371/journal.pone.0183578
Yassi F, Janetta B, Paul A, Ayesha NS (2017) MYC-Driven pathways in breast cancer subtypes. Biomolecules 7(3):53. https://doi.org/10.3390/biom7030053
Shanshan B, Subing C, Lianjin J, Margaret K et al. (2019) A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer. Oncogene 38(25):4977-4989. https://doi.org/10.1038/s41388-019-0768-8
Lina G, Jacob S, Angela G et al (2013) AR promotes ligand-independent prostate cancer progression through c-Myc upregulation. PLoS 8(5):e63563. https://doi.org/10.1371/journal.pone.0063563
Keely M, Nicole LM, Theresa EH, Hironobu S, Wayne DT (2014) Complexities of androgen receptor signalling in breast cancer. Endocr Relat Cancer 21(4): T161-81. https://doi.org/10.1530/ERC-14-0243
Jane LB, Stephen BF, Cheng H, Leticia C, Helen T, Suresh K, Peter RM, Adrian LH (2005) The AR is significantly associated with vascular endothelial growth factor and hypoxia sensing via hypoxia-inducible factors HIF-1a, HIF-2a, and the prolyl hydroxylases in human prostate cancer. Clin Cancer Res 11(21):7658-63. https://doi.org/10.1158/1078-0432.CCR-05-0460
Kurtis E, Gail F (2017) The AR and VEGF Mechanisms of androgen-regulated angiogenesis in prostate cancer. Cancers (Basel) 9(4):32. https://doi.org/10.3390/cancers9040032
Woodward WA, Wachsberger P, Burd R, Dicker AP (2005) Effects of androgen suppression and radiation on prostate cancer suggest a role for angiogenesis blockade. Prostate Cancer Prostatic Dis 8(2):127-32. https://doi.org/10.1038/sj.pcan.4500779
Qian N, Caigang L, Lei H, Min M, Xiaojin Z, Minna L, Shan S, Xiaoxiao Z, Xinhan Z (2013) Vascular endothelial growth factor receptor-1 activation promotes migration and invasion of breast cancer cells through epithelial-mesenchymal transition. PLoS One 8(6):e65217. https://doi.org/10.1371/journal.pone.0065217
Minna L, Lei H, Shan S, Shangke H, Du M, Lifeng L, Lu F, Peng X, Tianjie Q, Xinhan Z (2016) VEGF/NRP-1axis promotes progression of breast cancer via enhancement of epithelial-mesenchymal transition and activation of NF-κB and β-catenin. Cancer Lett 373(1):1-11. https://doi.org/10.1016/j.canlet.2016.01.010
Masoud N, Bagher, Keywan M (2019) Extracellular matrix (ECM) stiffness and degradation as cancer drivers. J Cell Biochem 120(3):2782-2790. https://doi.org/10.1002/jcb.27681
Saray Q, Rodrigo A, Enrique BV et al (2019) Role of matrix metalloproteinases in angiogenesis and cancer. Front Oncol 9:1370. https://doi.org/10.3389/fonc.2019.01370
Duffy M J, Maguire TM, Hill A, McDermott E, Higgins NO' (2000) Metalloproteinases: role in breast carcinogenesis, invasion and metastasis. Breast Cancer Res 2(4):252-7. https://doi.org/10.1186/bcr65
Cristian S, Ioana AB, Raluca C et al (2019) The role of matrix metalloproteinases in the epithelial-mesenchymal transition of hepatocellular carcinoma. Anal Cell Pathol (Amst) 2019:94239. https://doi.org/10.1155/2019/9423907
Arthur W, Sylvia A, Paolo M (2018) matrix metalloproteinase inhibitors in cancer therapy: Turning past failures into future successes. Mol Cancer Ther 17(6):1147-1155. https://doi.org/10.1158/1535-7163.MCT-17-0646.
Flavia M, Rocío Castillo-S, María JGet al (2020) Expression of metalloproteinases MMP-2 and MMP-9 is associated to the presence of AR in epithelial ovarian tumors. J Ovarian Res 13(1):86. https://doi.org/10.1186/s13048-020-00676-x
Yan Z, Yucheng S, Bin C, Aiting Y, Haoming J (2015) Elevated expression levels of androgen receptors and matrix metalloproteinase-2 and -9 in 30 cases of hepatocellular carcinoma compared with adjacent tissues as predictors of cancer invasion and staging. Exp Ther Med 9(3):905-908. https://doi.org/10.3892/etm.2014.2150
See-Tong P, Amilcar F, Lambert S, Yin-Choy C, Gunnar N, Ake P (2004) Regulation of matrix metalloproteinase 13 expression by androgen in prostate cancer. Oncol Rep 11(6):1187-92.
Luis O, Maria D, Julio V, Sara J, Rosario S, Ana C, Juan CR, Maria LL, Francisco JV (2008) AR expresion in breast cancer: relationship with clinicopathological characteristics of the tumors, prognosis, and expression of metalloproteases and their inhibitors. BMC Cancer 8:149. https://doi.org/10.1186/1471-2407-8-149
Masahiro M, Mina JB (2016) Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context. Oncotarget 7(22):31602-22. https://doi.org/10.18632/oncotarget.9395
Shreyas D, Bryan NB, F Michael H, Janet E (2009) Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway. BMC Cell Biol 10:94. https://doi.org/10.1186/1471-2121-10-94
Jing Y, Lin C, Jingshu X, Xue-Kang Q et al (2019) SHROOM2 inhibits tumor metastasis through RhoA-ROCK pathway-dependent and -independent mechanisms in nasopharyngeal carcinoma. Cell Death Dis 10(2):58. https://doi.org/10.1038/s41419-019-1325-7
Wen S, Shang Z, Zhu S, Chang C, Niu Y (2013) AR enhances entosis, a non-apoptotic cell death, through modulation of Rho/ROCK pathway in prostate cancer cells. Prostate 73:1306–1315. https://doi.org/10.1002/pros.22676
Kroiss A, Vincent S, Decaussin-Petrucci M, Meugnier E, Viallet J, Ruffion A, Chalmel F, Samarut J, Allioli N (2015) Androgen-regulated microRNA-135a decreases prostate cancer cell migration and invasion through downregulating ROCK1 and ROCK2. Oncogene 34(22):2846-55. https://doi.org/10.1038/onc.2014.222
Maria MM, Jorge E, Maria S et al (2016) Androgens regulate T47D cells motility and invasion through actin cytoskeleton remodeling. Front Endocrinol (Lausanne) 7:136. https://doi.org/10.3389/fendo.2016.00136

1Sulphorhodamineassay.pdf
Supp 1: Sulphorhodamine cytotoxicity assay illustrating the effect of different concentrations of EPI-001 (Table 1,2,3) or Enzalutamide (Table 4,5,6) on the absorbance of T47D cells as measured spectrophotometrically at 570 nm.
2Woundhealingassay.xlsx
Supp 2: Wound healing assay: data showing reduction in wound area after treatment with EPI-001 or Enzalutamide compared to control.
3ELISAcaliberationcurves.pdf
Supp 3: ELISA assay caliberation curves.
4ELISArawdata.xlsx
Supp 4: ELISA assay data for AR, ARV7, C-myc, NF-κB, E-Cadherin and N-Cadherin.
5ROCK1data.xlsx
Supp 5: ELISA assay data for ROCK1 enzyme.
6ROCK2data.xlsx
Supp 6: ELISA assay data for ROCK2 enzyme.
7Westernblot.pdf
Supp 7: Western blot bands for AR, ARV7, Cyclin E, CDK4, CDK6, Fibronectin, VEGF, MMP2 and MMP9 proteins.
8opticaldensitiesofWesternblot.xlsx
Supp 8: Data of optical densities of Western blotting.

Download PDF

Version 1

posted

You are reading this latest preprint version

Blocking Androgen Receptor/Androgen Receptor Variant7 Decreases Metastasis of Estrogen Receptor Positive Breast Cancer Cells Through Modulating Epithelial to Mesenchymal Transition

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell culture

Drug, chemicals & reagents

Sulphorhodamine-B (SRB) assay

Flowcytometry

Scratch wound healing assay

Immunocytostaining of ARV7

Enzyme-linked immunosorbent assay (ELISA)

Western blot analysis

Statistical analysis

Results

Effect of EPI-001 and Enzalutamide on the proliferation of T47D

EPI-001 and Enzalutamide induced S-phase cell cycle arrest and downregulated cell cycle regulatory proteins

EPI-001 and Enzalutamide inhibited cell migration and motility in T47D

Expression of AR/ARV7 in T47D and the effects of EPI-001 and Enzalutamide on their expression

EPI-001 downregulated NF-κB and C-myc protein levels in T47D

Effect of EPI-001 and Enzalutamide on E-Cadherin and N-Cadherin

EPI-001 and Enzalutamide downregulated Fibronectin

EPI-001 and Enzalutamide reduced angiogenesis

EPI-001 and Enzalutamide downregulated protein levels of matrix metalloproteinases (MMPs): MMP2 and MMP9

EPI-001 and Enzalutamide downregulated ROCK1 and ROCK2 levels

Discussion

Declarations

References

Supplementary Files

Status:

Version 1