Abnormal expression of dopamine receptors (DRs) has been described in multiple tumors, but their roles in breast cancer are inconclusive or contradictory since evidence of pro- and anti-tumoral effects have been reported. Herein, we analyzed the expression of DRs in breast cancer, especially in the subpopulation of cancer stem cells (CSCs), and evaluated the functional role of the receptors by pharmacological targeting.
Expression of DRD1, DRD2, DRD3, DRD4 and DRD5 was investigated in human breast tumors and cancer cell lines using public databases. Correlation between gene expression and clinical outcome was studied by Kaplan-Mayer analyses. By flow cytometry, we assessed DRD1, DRD2, and DRD4 expression in cultures of MCF-7 (luminal) and MDA-MB-231 (triple-negative) cells. Using the previously reported SORE6 reporter system we examined the differential expression of DRD1, DRD2, and DRD4 in CSCs and tumor-bulk cells. The effect of pharmacological modulation of DRs on stemness and cell migration was studied by quantification of the reporter-positive fraction and wound healing assays, respectively.
DRD1, DRD2 and DRD4 transcripts were expressed in breast tumors. DRD4 was overexpressed compared to normal tissue and showed prognostic value. DRD1, DRD2 and DRD4 transcripts were also found in MCF-7 and MDA-MB-231 cells, but only DRD1 and DRD4 proteins were detected. DRD4 was underexpressed in CSCs compared to tumor-bulk cells, whereas DRD1 was found only in the CSCs fraction, suggesting that those receptors may have relevance in stemness control. Subtoxic concentrations of DRD1-targeting compounds did not induced significant changes in the CSCs pool. On the other hand, DRD4 inhibition by Haloperidol slightly increased the CSCs content but also reduced cell migration.
Pharmacological modulation of DRD1 in MCF-7 or MDA-MB-231 cells seems to be irrelevant for stemness maintenance. DRD4 reduced expression in breast CSCs or its inhibition by Haloperidol favors CSCs-pool expansion. DRD4 inhibition can also reduce cell migration, indicating that DRD4 plays different roles in stem and non-stem breast cancer cells.
2022 Translational Cancer Research. All rights reserved.

Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists.
We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation.
Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with β1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC.
Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.
© 2020 Velázquez-Quesada et al.