Blockade of immune‐checkpoint programmed cell death protein 1 (PD‐1) or programmed cell death ligand 1 can enhance effector T‐cell responses. However, the lack of response in many patients to checkpoint‐inhibitor therapies emphasizes the need for combination immunotherapies to pursue maximal antitumor efficacy. We have previously demonstrated that antagonism of C‐X‐C chemokine receptor type 4 (CXCR4) by plerixafor (AMD3100) can decrease regulatory T (Treg)‐cell intratumoral infiltration. Therefore, a combination of these 2 therapies might increase antitumor effects. Here, we evaluated the antitumor efficacy of AMD3100 and anti‐PD‐1 (αPD‐1) antibody alone or in combination in an immunocompetent syngeneic mouse model of ovarian cancer. We found that AMD3100, a highly specific CXCR4 antagonist, directly down‐regulated the expression of both C‐X‐C motif chemokine 12 (CXCL12) and CXCR4 in vitro and in vivo in tumor cells. AMD3100 and αPD‐1 significantly inhibited tumor growth and prolonged the survival of tumor‐bearing mice when given as monotherapy. Combination of these 2 agents significantly enhanced antitumor effects compared with single‐agent administration. Benefits of tumor control and animal survival were associated with immunomodulation mediated by these 2 agents, which were characterized by increased effector T‐cell infiltration, increased effector T‐cell function, and increased memory T cells in tumor microenvironment. Intratumoral Treg cells were decreased, and conversion of Treg cells into T helper cells was increased by AMD3100 treatment. Intratumoral myeloid‐derived suppressor cells were decreased by the combined treatment, which was associated with decreased IL‐10 and IL‐6 in the ascites. Also, the combination therapy decreased suppressive leukocytes and facilitated M2‐to‐M1 macrophage polarization in the tumor. These results suggest that AMD3100 could be used to target the CXCR4‐CXCL12 axis to inhibit tumor growth and prevent multifaceted immunosuppression alone or in combination with αPD‐1 in ovarian cancer, which could be clinically relevant to patients with this disease.—Zeng, Y., Li, B., Liang, Y., Reeves, P. M., Qu, X., Ran, C., Liu, Q., Callahan, M. V., Sluder, A. E., Gelfand, J. A., Chen, H., Poznansky, M. C. Dual blockade of CXCL12‐CXCR4 and PD‐1‐PD‐L1 pathways prolongs survival of ovarian tumor‐bearing mice by prevention of immunosuppression in the tumor microenvironment. FASEB J. 33, 6596–6608 (2019). http://www.fasebj.org

Background Estrogen receptor β (ERβ) has been reported to play an anti-cancer role in breast cancer, but the regulatory mechanism by which ERβ exerts this effect is not clear. Claudin-6 (CLDN6), a tight junction protein, acts as a tumor suppressor gene in breast cancer. Our previous studies have found that 17β-estradiol (E2) induces CLDN6 expression and inhibits MCF-7 cell migration and invasion, but the underlying molecular mechanisms are still unclear. In this study, we aimed to investigate the role of ERβ in this process and the regulatory mechanisms involved. Methods Polymerase chain reaction (PCR) and western blot were used to characterize the effect of E2 on the expression of CLDN6 in breast cancer cells. Chromatin immunoprecipitation (ChIP) assays were carried out to confirm the interaction between ERβ and CLDN6. Dual luciferase reporter assays were used to detect the regulatory role of ERβ on the promoter activity of CLDN6. Wound healing and Transwell assays were used to examine the migration and invasion of breast cancer cells. Western blot, immunofluorescence and transmission electron microscopy (TEM) were performed to detect autophagy. Xenograft mouse models were used to explore the regulatory effect of the CLDN6-beclin1 axis on breast cancer metastasis. Immunohistochemistry (IHC) was used to detect ERβ/CLDN6/beclin1 expression in breast cancer patient samples. Results Here, E2 upregulated the expression of CLDN6, which was mediated by ERβ. ERβ regulated CLDN6 expression at the transcriptional level. ERβ inhibited the migration and invasion of breast cancer cells through CLDN6. Interestingly, this effect was associated with CLDN6-induced autophagy. CLDN6 positively regulated the expression of beclin1, which is a key regulator of autophagy. Beclin1 knockdown reversed CLDN6-induced autophagy and the inhibitory effect of CLDN6 on breast cancer metastasis. Moreover, ERβ and CLDN6 were positively correlated, and the expression of CLDN6 was positively correlated with beclin1 in breast cancer tissues. Conclusion Overall, this is the first study to demonstrate that the inhibitory effect of ERβ on the migration and invasion of breast cancer cells was mediated by CLDN6, which induced the beclin1-dependent autophagic cascade. Electronic supplementary material The online version of this article (10.1186/s13046-019-1359-9) contains supplementary material, which is available to authorized users.

Supplemental Digital Content is available in the text

The Wnt/β-catenin pathway is constitutively active and promotes multiple tumor processes, including breast cancer metastasis. However, the underlying mechanism by which the Wnt/β-catenin pathway is constitutively activated in breast cancer metastasis remains unclear. Inhibition of Wnt antagonists is important for Wnt/β-catenin signaling activation, and post-transcriptional regulation of these antagonists by microRNAs (miRNAs) might be a possible mechanism underlying signaling activation. Regulation of nuclear pre-mRNA domain-containing 1A (RPRD1A) is a known inhibitor of cell growth and Wnt/β-catenin signaling activity, but the function and regulatory mechanism of RPRD1A in breast cancer have not been clarified. The aim of this study was to understand how regulators of the Wnt/β-catenin pathway may play a role in the metastasis of this cancer. Methods: RPRD1A expression and its association with multiple clinicopathological characteristics was analyzed immunohistochemically in human breast cancer specimens. miR-454-3p expression was analyzed using real-time PCR. RPRD1A or miR-454-3p knockdown and overexpression were used to determine the underlying mechanism of their functions in breast cancer cells. Xenografted tumor model, 3D invasive culture, cell migration and invasion assays and sphere formation assay were used to determine the biofunction of RPRD1A and miR-454-3p in breast cancer. Electrophoretic mobility shift assay (EMSA), luciferase reporter assay, and RNA immunoprecipitation (RIP) were performed to study the regulation and underlying mechanisms of RPRD1A and miR-454-3p expression and their correlation with the Wnt/β-catenin pathway in breast cancer. Results: The Wnt/β-catenin signaling antagonist RPRD1A was downregulated and its upstream regulator miR-454-3p was amplified and overexpressed in metastatic breast cancer, and both were correlated with overall and relapse-free survival in breast cancer patients. The suppression by miR-454-3p on RPRD1A was found to activate Wnt/β-catenin signaling, thereby promoting metastasis. Simultaneously, three other negative regulators of the Wnt/β-catenin pathway, namely, AXIN2, dickkopf WNT signaling pathway inhibitor (DKK) 3 and secreted frizzled related protein (SFRP) 1, were also found to be targets of miR-454-3p and were involved in the signaling activation. miR-454-3p was found to be involved in early metastatic processes and to promote the stemness of breast cancer cells and early relapse under both in vitro and in vivo conditions. Conclusions: The findings indicate that miR-454-3p-mediated suppression of Wnt/β-catenin antagonist RPRD1A, as well as AXIN2, DKK3 and SFRP1, sustains the constitutive activation of Wnt/β-catenin signaling; thus, miR-454-3p and RPRD1A might be potential diagnostic and therapeutic targets for breast cancer metastasis.