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.