Enzalutamide and EPI-001 in T47D: Blocking Androgen receptor/Androgen receptor variant7 modulates NF-ĸB/c-Myc axis, cyclins A, E, &amp; C, epithelial to mesenchymal transition markers, angiogenesis, and metastasis

doi:10.21203/rs.3.rs-2311551/v1

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Enzalutamide and EPI-001 in T47D: Blocking Androgen receptor/Androgen receptor variant7 modulates NF-ĸB/c-Myc axis, cyclins A, E, & C, epithelial to mesenchymal transition markers, angiogenesis, and metastasis

https://doi.org/10.21203/rs.3.rs-2311551/v1

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Androgen receptor (AR) and its splicing variant 7 (ARv7) play vital roles in the pathobiology of breast cancer (BC) but their role in the estrogen receptor-positive (ER+) type is controversial, hence, we studied the influence of the blockers of AR (Enzalutamide) and ARv7 (EPI-001) on tumorigenesis processes using T47D, an ER+ BC cell line. We showed that although both inhibitors failed to reduce cell growth and affect AR content, only Enzalutamide reduced the ARv7. Mechanistically, the drugs successfully arrested the cell cycle at S-phase and downregulated the protein expression of cyclins A, E, & C. Additionally, they inhibited the cell proliferation stimulator nuclear factor kappa B (NF-ĸB), whereas only EPI-001 reduced the cell regulatory marker c-Myc. They also opposed the endothelial-to-mesenchymal transition (EMT) process, by boosting the epithelial marker E-cadherin and reducing the protein expression of the mesenchymal marker fibronectin. Their anti-metastatic potential was evidenced by the hindrance of cell migration using the wound healing assay and further confirmed by the downregulation of metalloproteinase (MMP) 2 and 9 protein expression, and protein content of Rho kinase (ROCK)1 and 2. Besides, by downregulating the protein expression of vascular endothelial growth factor (VEGF) the drugs point to their anti-angiogenic aptitude. In conclusion, this in-vitro study is the first to prove the importance of blocking AR/ARv7 using Enzalutamide and EPI-001 in decreasing cancer cell survival, EMT, and metastasis in ER+ BC cells, findings that still need further studies to unveil the role of these inhibitors in BC.

Androgen receptor

Androgen receptor variant 7

metastasis

Epithelial to mesenchymal transition

breast cancer

Androgen receptor (AR) is a vital element in the pathogenesis of breast cancer (BC), the most common malignancy among women worldwide (1). AR is expressed in more than 60% of BCs and up to 90% of estrogen receptor (ER) + tumors, signifying possible androgen responsiveness (2), however this role is controversial and still far from clarity which restrains the application of AR-directed therapies.

AR performs a tumor suppressor role in ER + BC (3) by binding to a group of estrogen response elements, thereby preventing the activation of target genes that facilitate the stimulatory effects of 17-β-estradiol on BC cells (4). Additionally, blocking AR stopped the estradiol-induced proliferation of an ER+/AR + cell line model (5) and suppressed the proliferation of MCF7cells (6). In the same milieu, constitutively active AR sustained the metastasis of endocrine-resistant hormone receptor-positive BC (7). However, other researchers suggest the oncogenic impact of AR and its potential role in the acquired resistance against ER + treatments for BC (8).

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 which produce constitutively active, ligand-independent, transcription factors or variants resistant to drugs that reduce androgen production and biosynthesis (9). 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 (10). Moreover, their role in BC has been recognized and is considered an area of active research (11).

The role of aberrant expression of AR/ARv7 in the progression of different cancers, including breast (11) and prostate (12) cancers, has been evaluated by endorsing tumor cell growth, however, the impact of AR/ARv7 on cell proliferation, epithelial to mesenchymal transition (EMT) process and metastasis was scarcely appraised. EMT is implicated in carcinogenesis and provides metastatic properties to cancer cells (13) and the EMT-derived tumor cells attain stem cell characteristics and display a noticeable therapeutic resistance (14).

Accordingly, we assessed the role of AR and ARv7 blockers using Enzalutamide and EPI-001, respectively, on cell cycle, EMT, and metastasis using T47D.

2.1. Cell culture

T47D cells were obtained from the American Type Culture Collection (ATCC, MN, USA) and were maintained and routinely checked at the Tissue culture facility of the Department of Cancer Biology, Unit of Pharmacology and Experimental Therapeutics, National Cancer Institute (NCI), Cairo University, Egypt. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), L-glutamine (2 mM), sodium bicarbonate (1.5 g/L), and penicillin/streptomycin (1%) and kept in a humidified atmosphere with 5% CO₂ at 37°C.

2.2. Ethical statement

The present study was conducted only on T47D (ER+ breast cancer) cell line obtained from the American Type Culture Collection (ATCC, MN, USA) and did not include any human subjects or experimental animals. The methods comply with pertinent guidelines and regulations and the protocol was approved by the Committee for the Safe Handling and Disposal of Chemical and Biological Substances affiliated with the Research Ethics Committee of the Faculty of Pharmacy at Cairo University (Cairo, Egypt; PT 2126).

2.3. Sulphorhodamine-B (SRB) assay

The antitumor activity of Enzalutamide and EPI-001(ApexBio, TX, USA) on T47D cells were evaluated by the SRB assay. Briefly, cells were seeded for 24 hr in a 96-well plate at a density of 3×10³ cells per well and treated with different concentrations (6.25, 12.5, 25, 50, 100, and 200 µM) of Enzalutamide or EpI-001. Drugs were dissolved in dimethylsulfoxide to prepare a stock solution of 1 mM. For each concentration, two wells were used to be 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 an ELISA microplate reader (TECAN sunrise, Germany). The cell survival fraction was calculated by dividing the O.D. of treated cells over that of control cells.

N.B. For 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 being calculated based on the inhibition of AR and ARv7 (5, 15), since we were not able to determine the IC50 for either drug using the SRB test.

2.5. Immunocytostaining of ARv7

T47D cells were collected/washed with PBS, resuspended in DMEM, and placed on a glass slide incubated in H₂O₂ for 15 min. then washed again using PBS to be incubated with a monoclonal antibody against ARv7 for an hr. Afterward, a biotinylated secondary antibody was added, incubated at room temperature for 10 min, then washed with PBS, and 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.

2.6. Determination of cell cycle phase using 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. Consequently, 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 an EPICS® C Flow cytometer (FLA, USA).

2.7. 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 a 1 ml pipette tip through each well center in one direction. The wells were washed to remove any detached cells, then the cells were treated with Enzalutamide or EPI-001 for 24 hr to be fixed with 3.7 % paraformaldehyde for 30 min. Finally, the cells were stained with 1 % crystal violet for 30 min. The stained monolayer was photographed and the reduction in gap area was measured (ImageJ software, MD, USA).

2.8. Assessment of protein content of AR, ARv7, NF-κB, c-Myc, E-Cadherin, N-Cadherin, ROCK1, and ROCK2 using the ELISA technique

The ELISA kits were obtained from Innova Biotectech (BJ, China) for AR (cat #: In-Hu4116), ARv7 (cat #: In-Hu4117), c-Myc (cat #: In-Hu1853), NF-κB (cat #: In-Hu2637), E-Cadherin (cat #: In-Hu1892), and N-Cadherin (cat #: In-Hu4118), whereas for ROCK1 (Catalog #: OKEH06554) and ROCK2 (catalog #: LS-F22011) the ELISA kits were purchased from Aviva Systems Biology, (LA, USA) and LifeSpan BioSciences, (WA, USA), respectively. The cells were harvested and their protein was quantified using Bradford assay kit (Pierce, Rockford, IL, USA). ELISA quantification was carried out in accordance with the manufacturer’s instructions and the protein contents were calculated from the constructed calibration curves using second-order polynomial curve-fitting models.

2.9. Determination of AR, ARv7, cyclins A, C, and E, fibronectin, MMP2 & 9, and VEGF protein expression using Western blot analysis

The primary antibodies used for AR (cat #: sc-7305), cyclins A (cat #: sc-271682), cyclin E (cat #: sc-377100), fibronectin (cat #: sc-8422), matrix metalloproteinase (MMP)2 (cat #: sc-13595), MMP9 (cat #: sc-393859), and VEGF (cat #: sc-7269) were purchased from Santa Cruz Biotechnology, (TX, USA), whereas those for cyclin C (catalog #: 26464-1-AP) and ARv7 (catalog #: AG10008) were procured from Proteintech (IL, USA) and A&G Precision Antibody (MD, US), respectively. The Ready Prep^TMprotein extraction kit (Bio-Rad Inc., CA, USA) was used to extract protein from the pellets obtained from the collected cells and protein analysis was conducted using the Bradford assay kit. The extracted proteins were separated by SDS-PAGE using TGX Stain-Free™ FastCast™ (Bio-Rad Inc., CA, USA) and blotted onto PVDF membranes that were blocked with 5% non-fat dry milk and incubated at 4°C with primary antibodies overnight followed by incubation with peroxidase-conjugated secondary antibodies. The β-actin antibody was probed in the membrane for normalization. The intensities of the bands were normalized against β-actin and were analyzed on the ChemiDoc MP imager.

2.10. Statistical analysis

The data were presented as mean ± S.E.M and statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (p ≤ 0.05) and F values were expressed to indicate the ratio between and within group variances using IBM SPSS Statistics for Windows, Version 26.0 (NY, USA). Graphs were sketched using GraphPad InStat, version 5.0 (LA, USA).

3.1. Screening the protein expression of ARV/ARV7 and determination of IC50 of their inhibitors in T47D cells

Fig 1 reveals the protein expression of ARv7 in the T47D cells using the (a) immunocytochemical and (b) Western blot examinations. The latter technique shows also the expression of AR in this type of BC cell line. Using the SRB assay, no significant inhibition in the proliferation of T47D cells was observed following treatment by either (c) EPI-001 or (d) Enzalutamide. For the two blockers, no IC50 value was detected.

3.2. EPI-001 and Enzalutamide arrested cell cycle at S-phase and downregulated cell cycle regulatory proteins

As shown in Fig 2 (a & b), 56.43% and 24.72% of the cell population were retained in the S-phase after treatment with EPI-001 and Enzalutamide, respectively as compared to 4.36% in the untreated control group. Furthermore, they caused (c & d) a sharp downregulation in the assessed cyclins A [F (2,8) = 163.35, P <0.001], E [F (2,8) = 352.8, P <0.00)], and C [F (2,8) = 123.11, P <0.001], compared to the untreated control cells.

3.3. Effect of EPI-001 and Enzalutamide on the protein content of AR, ARv7, NF-κB and c-Myc in T47D cells

Regarding the effect of the two inhibitors on the protein content of AR and ARv7, Fig 3 (a & b) reveals that treatment of the T47D cells with EPI-001 failed to alter the protein content of either the androgen receptor or its variant, as compared to the untreated control cells. However, treatment with Enzalutamide tended to elevate the protein content of the AR and, unexpectedly, inhibited the protein content of its tested variant ARv7 significantly compared to the EPI-001 treated group [F (2,11) = 8.724, P =0.008)]. On the other hand, both inhibitors succeeded to affect the NF-ĸB/c-Myc axis. As illustrated in Fig 3 (c & d), EPI-001 halved the protein expression of the transcription factor NF-ĸB [F (2,11) = 15.035, P =0.001] which is a commonly observed phenomenon in BC, whereas the effect of Enzalutamide was less pronounced. Regarding c-Myc, a key regulator of cell growth, and apoptosis, it was significantly reduced by EPI-001 [F (2,11) = 211.097, P <0.001] compared to the control group, whereas treatment with the AR blocker Enzalutamide displayed no significant effect relative to the control group.

3.4. Effect of EPI-001 and Enzalutamide on EMT-related markers in T47D cells

As depicted in Fig 4, treatment with both EPI-001and Enzalutamide mired the EMT process; this was confirmed by the boosted protein content of the (a) epithelial marker E-cadherin compared to the control group, [F (2,11) = 468.232, P <0.001]. Although the two treatments did not alter the protein content of the (b) mesenchymal marker N-Cadherin, relative to the untreated control, they succeeded to deter the (c & d) protein expression of fibronectin, another mesenchymal marker. Both EPI-001 and Enzalutamide significantly reduced fibronectin [F (2,8) = 70.39, P <0.001] by 70.67% and 68% relative to the control group, respectively.

3.5. EPI-001 and Enzalutamide inhibited cell migration and angiogenesis in T47D

The ability of either inhibitor to hinder cell migration is illustrated in Fig 5 (a & b). The scratch area revealed a reduction of 61% in the control group after 24 hr; however, the scratch gap was widened in the treated groups, where the cellular migration was further reduced in the EPI-001 and Enzalutamide treated groups significantly to 33.67% and 34.54%, respectively as compared to control group after 24 hr [F (2,17) = 44.358, P <0.001]. The effect of the two inhibitors was additionally assured biochemically (Fig 5 c &d) where both EPI-001 and Enzalutamide hampered cell migration by significantly downregulating the protein expression of MMP2 [F (2,8) = 51.79, p<0.001] and MMP9 [F (2,8) = 152.73, P <0.001]. EPI-001 significantly reduced MMP2 and MMP9 by 66.5% and 69.79%, respectively and Enzalutamide downregulated the protein expression of MMP2 and MMP9 by 67.77% and 66.84% respectively relative to their control values. Moreover, treatment of T47D cells with EPI-001 and Enzalutamide downregulated the angiogenic marker VEGF band densities by 72.21% and 75.49%, respectively [F (2,8) = 82.74, P <0.001] relative to the control group.

3.6. EPI-001 and Enzalutamide reduced ROCK1 and ROCK2

The impact of AR and ARv7 blockage on Rho kinase 1 and 2 (ROCK1 & 2), which play a role in adhesion, migration, proliferation, and apoptosis, was evaluated and displayed in Fig 6 (a & b). A marked reduction of ROCK1 and ROCK2 contents was observed when the breast cancer cells were treated with EPI-001 and Enzalutamide. ROCK1: [F (2,11) = 335.26, P <0.001]; ROCK2: [F (2,11) = 364.663, (P <0.001)].

Although the expression of AR in breast cancer has appointed it an attractive therapeutic target, its role in breast cancer is still controversial since it can either inhibit or promote breast tumor growth and metastasis (3, 8). Among the AR variants, ARv7 is drawing attention as a prospective marker for one of the resistance mechanisms to anti-AR therapy in castration-resistant prostate cancer patients (CRPC) (12) and has also been recently detected in breast cancer subtypes (11). However, the role of both AR and ARv7 in ER+ breast cancer needs further clarification.

It is well documented that the tumorigenesis events of cancer instigation and advancement involve several correlated steps including tumor growth, angiogenesis, neovascularization, invasion, and metastasis. Here, we have evaluated the impact of inhibiting AR and ARv7 on the tumorigenesis multi-steps using an ER+ breast cancer cell line. Studying the cytotoxicity of the tested drugs, we showed that neither drug was able to affect the cell survival/proliferation of the ER+ cell line T47D. Although this was not expected, another study supported our findings, where Caiazza and co-workers (2016) reported that the sensitivity of breast cancer proliferation to treatment with enzalutamide was widely variable with a marginal effect of enzalutamide on the T47D cell line (IC50 >50 µM) (16).

Nevertheless, on the molecular level, both drugs were able to affect growth-related biomarkers; the first is the transcription factor NF-κB, which is activated in some types of cancer cells and possesses pleiotropic roles in different aspects of tumorigenesis, such as enhancing cancer-cell proliferation, increasing angiogenesis, and enhancing metastatic potential (17). The link between NF-κB and AR was recounted previously, where the promoter region of the AR gene possesses a NF-κB response element and it was found that the stimulation of the latter increases AR in prostate cancer cells (18). These results, hence, nominate NF-κB to be a target for breast cancer therapy through the regulation of AR. In line with this notion, our results showed that the content of NF-κB was reduced in the T47D-treated cells by either of the used inhibitors and can be partly responsible for the decrease of its downstream molecules.

The first downstream target(s) evaluated here are cyclins A, E, and C, which play important roles in breast cancer and regulate cell cycles. In our study treatment of T47D cells with either Enzalutamide or EPI-001 succeeded in markedly downregulating the protein expressions of the tested cyclins. The inhibited cyclins were accompanied by a decrease in the number of cells in the G1 phase and cell cycle arrest in the S-phase, results that are in harmony with a previous study which indicated that the blockade of AR signaling decreased the G1-S cyclins in a resistant breast cancer cell line (19). Although it is not clear how the effect on the cell cycle was not translated into a decrease in cell proliferation, this can be explained by the report of Sherr and Roberts (20), who in their review argued about the necessity of cyclins D and E and their Cyclin-dependent kinases (CDKs) in cell survival and stated that despite the disruption of the genes encoding them in the mouse germ line, much of fetal development occurs normally in their absence. Other researchers also underlined the existence of an 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 (21).

Another down target of NF-ĸB is c-Myc which also acts as a transcription factor for a miscellaneous array of genes to regulate the progression of the cell cycle, cell growth, and apoptosis, to have a proliferative task and to impart resistance to chemotherapeutic agents in ER+ breast cancer cell lines (22). The link between c-Myc and AR has been stated earlier, where c-Myc has a critical role in regulating the expression of AR and AR variants in CRPC and through the upregulation of c-Myc, AR promotes ligand-independent prostate cancer progression (23). Moreover, it was reported that AR cooperates with c-Myc to induce tumorigenesis (24) and that the levels of c-Myc correlate positively with ARv7 in prostate cancer (23). Despite these verities, our results showed that c-Myc was inhibited only with the variant inhibitor but not by enzalutamide. Indeed, a recent study reported that the use of a Myc inhibitor has synthesized the prostate cancer cells to enzalutamide to inhibit cell viability (22), a finding that may highlight the necessity of another factor to help enzalutamide inhibit c-Myc.

Aside from its regulatory role on cell growth, the overexpression of c-Myc was reported to trigger the EMT process in triple-negative MDA-MB-231 cells (14). Breast cancer cells undergo EMT modifications, a process that aids in enhancing tumor progression, dissemination, metastasis, and therapy resistance (14). This process is also under the control of the upstream transcription factor NF-κB, which was found to modulate EMT in breast cancer via controlling a number of markers involved in EMT as cadherins and fibronectin (25) and is associated with high ER expression, invasion, and metastasis (26). Although, the implication of AR and AR splice variant 7 on the process of EMT in prostate cancer was assessed before, however, their impact on ER+ (T47D) breast cancer cells was first evaluated herein by measuring the contents of E-cadherin and N-cadherin, and the protein expression of fibronectin. Our data showed that the receptor blocker and the variant inhibitor have boosted the content of the epithelial marker E-cadherin to strengthen cell-cell adhesion and hinder the invasion and metastasis of the tumor, an effect that can be related to the inhibited NF-ĸB which plays a role in the regulation of E-cadherin (25). Despite their ability to suppress the protein expression of the mesenchymal marker fibronectin, the tested inhibitors failed to alter the content of the second mesenchymal marker N-Cadherin. Indeed, the decrease in E-cadherin and the increase in N-cadherin correlate with increased invasiveness and metastasis of tumors, where N-cadherin is regarded as an oncoprotein that stimulates invasion, proliferation, angiogenesis, and metastasis (27). Similar to N-cadherin, fibronectin glycoprotein is associated with metastatic tumors and poor prognosis in many cancers including breast cancer (28). In 2008, Liu et al. (29) 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, results that can support our results. In prostate cancer cells, it was also reported that the constitutively active AR variants upregulate the expression of several mesenchymal markers including N-cadherin (30). Later, Das et al. (31) identified a novel pathway in prostate cancer whereby miR-1207-3p regulates the AR via a pathway involving fibronectin.

Our study also evaluated the anti-angiogenic potential of the two inhibitors in the present breast cell line and our data documented the aptitude of both drugs to sharply downregulate the protein expression of the angiogenic marker VEGF. Once more, the decreased content of NF-ĸB by the two treatments can be one reason behind the severely downregulated VEGF being one of its downstream targets (32). In line with the present data, a close tether between the angiogenic cytokine VEGF and AR in prostatic tumors has been identified; where 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 (33). Moreover, it has been suggested that alterations in cellular pathways related to both endocrine and VEGF may contribute to breast cancer progression through EMT. Therefore, in the present study, it is possible that the tested blockers modulated EMT by downregulating the protein expression of VEGF.

Besides its known angiogenic role, VEGF partakes in tumor metastasis, where it shares in the breakage of ECM by enhancing the release of the proteolytic enzymes MMP2 and MMP9 from tumor cells (34). These enzymes are implicated also in angiogenesis, invasion and metastasis in various tumors including breast cancer (35). Moreover, elevated levels of MMPs in ECM significantly increase EMT (36), and their expression was associated with the presence of AR in epithelial ovarian tumors and hepatocellular carcinoma to be one risk factor for overall survival or predictive of invasion and metastasis (37).

In the same context, it was reported that the androgen inhibitor Bicalutamide was able to abolish MMPs in prostate cancer (38). Despite breast cancer exhibiting a highly metastatic potential, a few studies have been performed to evaluate AR expression in primary tumors and metastatic samples. A previous study revealed that in breast cancer patients, the expression of some MMPs was higher in AR-positive tumors than in AR-negative ones (39). Further, androgens were reported to regulate the invasion and motility of the T47D cells (40). On the other hand, the ARv7 was recounted to be linked with metastasis in the case of CRPC (12). These studies hence, concur with our results in which the anti-metastatic effect of the two blockers Enzalutamide and EPI-001 was evidenced by the curbed protein expression of MMP2 and MMP9 in T47D cells and the widened gap in the Scratch wound healing assay to concur with a previous study which found that Enzalutamide inhibited cell migration and invasion in TNBC cell line in an AR-dependent manner (16). In the same regard, the possible role of NF-κB cannot be ignored since a previous study has suggested an association between high ER expression, invasion, metastasis, and NF-κB (41); thus, the inhibitory effect of the current drugs on NF-κB may be implicated, at least in part, in decreasing metastasis in ER+ breast cancer.

The last markers assessed herein are ROCK1 and ROCK2 which intermingle with the above markers and play a role in regulating several tumorigenic processes including tumor cell invasion, and metastasis. For example, the ROCK pathway has been involved in regulating EMT in renal and nasopharyngeal carcinoma cells (42), and by causing cytoskeletal rearrangement, it plays a central role in breast cancer cells and their disrupted tissue architecture and thereby therapy targeting the ROCK signaling cascade may provide a therapeutic opportunity for breast cancer (43). Moreover, links have been reported between androgens, AR, and ROCK pathways (44), where androgens induce the remodeling of the actin cytoskeleton by regulating moesin expression and activation, causing enhanced endothelial cell migration in hUVECs cells (45). Consequently, in our work, the AR inhibitors Enzalutamide and EPI-001 caused a marked reduction in the protein contents of ROCK1 and ROCK2 in T47D cells to further support their anti-metastatic effect. Moreover, the inhibition of ROCKs can participate in reducing cell proliferation by reducing c-Myc which was affected herein by the variant inhibitor only. A previous study has highlighted the potential antiproliferative role of ROCK inhibitors by preventing the transcriptional activity of the oncogenic marker c-Myc in breast cancer (14).

Based on these findings, our study indicated that blocking AR/ARv7 by Enzalutamide or EPI-001 decreased markers of proliferation (NF-ĸB, c-Myc), cell cycle (Cyclins A, C, E), EMT (E-cadherin, fibronectin), angiogenesis (VEGF), and hence metastasis (MMP2, 9, ROCK1 and 2) in ER+ breast cancer cells. Despite the inhibition of most of the tested markers, few were not affected as expected, findings that necessitate further preclinical and clinical studies to unveil this controversy which may have strong clinical benefits in the future.

Declaration/Funding

This work was supported by Cairo University, Grant number 170

Competing interests

The authors declare no competing financial and/or non-financial interests that can be perceived to influence the results and/or discussion reported in this paper

Consent to participate

Not applicable.

Author contribution

BAM: methodology, analysis, investigation, drafting

HSE: design, supervision, review

GM: methodology

MS: methodology

SS: design, data analysis, supervision, review

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

Conflict of interest

None

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Enzalutamide and EPI-001 in T47D: Blocking Androgen receptor/Androgen receptor variant7 modulates NF-ĸB/c-Myc axis, cyclins A, E, & C, epithelial to mesenchymal transition markers, angiogenesis, and metastasis

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