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The tumor microenvironment contributes to tumor progression and metastasis. Cancer-associated fibroblasts (CAFs) form a major cellular component of the tumor microenvironment. In this study, we further explored the mechanisms underlying the tumor-promoting roles of CAFs. Methods: Patient-derived CAFs and normal fibroblasts (NFs) were isolated from breast carcinomas and adjacent normal breast tissue. Exosomes were isolated by ultracentrifugation and CAF-derived exosomal microRNAs were screened using next-generation sequencing technology. MiR-500a-5p expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization; Tumor cell proliferation was determined by MTT assays and three-dimensioned (3D) cultures, and tumor metastasis was determined by Transwell assays in vitro . In vivo assays were performed in a nude mouse subcutaneous xenograft model. Results: We confirmed that CAF-derived exosomes significantly promoted the proliferation and metastasis of breast cancer cells. MiR-500a-5p was highly expressed in MDA-MB-231 and MCF7 cells treated with CAF-derived exosomes. The upregulation of miR-500a-5p was also confirmed in CAFs and CAF-derived exosomes. MiR-500a-5p was transferred from CAFs to the cancer cells, and subsequently promoted proliferation and metastasis by binding to ubiquitin-specific peptidase 28 (USP28). Conclusions: The present study demonstrates that CAFs promote breast cancer progression and metastasis via exosomal miR-500a-5p and indicate that inhibiting CAF-derived miR-500a-5p is an alternative modality for the treatment of breast cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Tamoxifen is the cornerstone of adjuvant therapy for hormone receptor-positive breast cancer. Despite its efficacy, limited drug sensitivity and endocrine resistance remain the important clinical challenges. The main objective of this study was to investigate fatostatin, which was found to sensitize breast cancer to the antitumour effect of tamoxifen both in vitro and in vivo., Methods: Fatostatin-induced ER degradation was detected by immunoprecipitation assay. The antitumour effect of fatostatin and tamoxifen on MCF-7 and T47D cells was assessed by MTT and colony forming assays. Cell cycle arrest was detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and TUNEL assay. Autophagy was detected by MDC assay and acridine orange staining. Migration and invasion assays were performed using a Transwell system, and the efficacy of the synergistic use of fatostatin and tamoxifen in vivo was evaluated using an MCF-7 xenograft model in BALB/c nu/nu female mice., Results: The synergistic use of fatostatin and tamoxifen significantly suppressed cell viability and invasion, induced cell cycle arrest, and regulated apoptosis and autophagy in MCF-7 and T47D cell lines via PI3K-AKT-mTOR signalling. Additionally, the expression levels of Atg7/12/13, beclin and LC3B increased while p-mTOR and P62 expression levels decreased after treatment with fatostatin and tamoxifen. Tumor growth in the xenograft model was suppressed significantly with the synergistic treatment of fatostatin and tamoxifen., Conclusion: Fatostatin could induce ER degradation by K48-linked polyubiquitination, which was the key mechanism contributing to tamoxifen inhibition of PI3K-AKT-mTOR signalling in breast cancer. Fatostatin may have a promising clinical use for ER-positive breast cancer patients., Competing Interests: The authors declare no conflicts of interest for this work., (© 2020 Liu et al.)

Background: Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown., Methods: Based on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1., Results: LncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter., Conclusions: Collectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer.

Tamoxifen (TAM) is frequently used to treat patients with estrogen receptor-positive (ER+) breast cancer; however, the development of endocrine resistance represents a major impediment for successful treatment. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) may serve critical roles in regulating endocrine resistance in breast cancer. In the present study, it was determined that the expression of lncRNA in nonhomologous end joining pathway 1 (LINP1) was increased in tamoxifen-resistant breast cancer cells, and that LINP1 knockdown significantly attenuated the tamoxifen resistance and viability of tamoxifen-resistant breast cancer cells in vitro and in vivo. LINP1 knockdown increased apoptosis in cells following treatment with tamoxifen. Furthermore, LINP1 overexpression resulted in increased cell mobility by regulating the EMT process. Mechanistically, LINP1 is a direct target of ER-mediated transcriptional repression, and both tamoxifen treatment and hormone deprivation increased the expression of LINP1. LINP1 overexpression was associated with downregulation of the levels of ER protein and attenuated the estrogen response, which is a pivotal contributing factor to anti-estrogen resistance. Taken together, the present study highlights the pivotal role of LINP1 in tamoxifen resistance, which may serve as a potential target to improve the effectiveness and efficacy of tamoxifen treatment in breast cancer., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Breast cancer is one of the most common malignancies among women throughout the world and is the major cause of most cancer-related deaths. Several explanations account for the high rate of mortality of breast cancer, and metastasis to vital organs is identified as the principal cause. Over the past few years, intensive efforts have demonstrated that breast cancer exhibits metastatic heterogeneity with distinct metastatic precedence to various organs, giving rise to differences in prognoses and responses to therapy in breast cancer patients. Bone, lung, liver, and brain are generally accepted as the primary target sites of breast cancer metastasis. However, the underlying molecular mechanism of metastatic heterogeneity of breast cancer remains to be further elucidated. Recently, the advent of novel genomic and pathologic approaches as well as technological breakthroughs in imaging analysis and animal modelling have yielded an unprecedented change in our understanding of the heterogeneity of breast cancer metastasis and provided novel insight for establishing more effective therapeutics. This review summarizes recent molecular mechanisms and emerging concepts on the metastatic heterogeneity of breast cancer and discusses the potential of identifying specific molecules against tumor cells or tumor microenvironments to thwart the development of metastatic disease and improve the prognosis of breast cancer patients., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Increasing evidence has indicated that circular RNAs (circRNAs) play a critical role in cancer development. However, only a small number of circRNAs have been experimentally validated and functionally annotated. In this study, using a high-throughput microarray assay, we identified a novel circRNA, circKDM4C, which was downregulated in breast cancer tissues with metastasis. Furthermore, we analyzed a cohort of breast cancer patients and found that circKDM4C expression was decreased in breast cancer tissues, and lower circKDM4C expression was associated with poor prognosis and metastasis in breast cancer. Functionally, we demonstrated that circKDM4C significantly repressed breast cancer proliferation, metastasis, and doxorubicin resistance in vitro and in vivo. Mechanistically, using a dual-luciferase activity assay and AGO2 RNA immunoprecipitation, circKDM4C was identified as a miR-548p sponge. We also found that PBLD was a direct target of miR-548p, which functioned as a tumor suppressor in breast cancer. Moreover, miR-548p overexpression was able to reverse the circKDM4C-induced attenuation of malignant phenotypes and elevated expression of PBLD in breast cancer cells. Taken together, our data indicate that circKDM4C might have considerable potential as a prognostic biomarker in breast cancer, and support the notion that therapeutic targeting of circKDM4C/miR-548p/PBLD axis may be a promising treatment approach for breast cancer patients.

Tamoxifen is the most commonly used endocrine therapy for patients with hormone receptor (HR)-positive breast cancer. Despite its initial therapeutic efficacy, many patients eventually develop drug resistance, which remains a serious clinical challenge. To investigate roles of circular RNAs (circRNAs) in tamoxifen resistance, a tamoxifen-resistant MCF-7 cell line was established and screened for its circRNA expression profile by RNA sequencing. hsa_circ_0025202, a circRNA that was significantly downregulated, was selected for further investigation. Using a large cohort of clinical specimens, we found that hsa_circ_0025202 exhibited low expression in cancer tissues and was negatively correlated with lymphatic metastasis and histological grade. Gain- and loss-of-function assays indicated that hsa_circ_0025202 could inhibit cell proliferation, colony formation, and migration and increase cell apoptosis and sensitivity to tamoxifen. Bioinformatics and luciferase reporter assays verified that hsa_circ_0025202 could act as a miRNA sponge for miR-182-5p and further regulate the expression and activity of FOXO3a. Functional studies revealed that tumor inhibition and tamoxifen sensitization effects of hsa_circ_0025202 were achieved via the miR-182-5p/FOXO3a axis. Moreover, in vivo experiments confirmed that hsa_circ_0025202 could suppress tumor growth and enhance tamoxifen efficacy. Taken together, hsa_circ_0025202 served an anti-oncogenic role in HR-positive breast cancer, and it could be exploited as a novel marker for tamoxifen-resistant breast cancer., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Recent studies have shown that long non-coding RNAs (lncRNAs) are involved in a number of biological processes; however, further study is still warranted to comprehensively reveal their functions. In this study, we showed that the lncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) was related to breast cancer cell proliferation, metastasis and chemoresistance. Loss- and gain-of function studies were used to assess the role of LINP1 in promoting breast cancer progression. LINP1 knockdown mitigated breast cancer cell growth by inducing G1-phase cell cycle arrest and apoptosis. LINP1 also promoted breast cancer cell metastasis and influenced the expression of epithelial-mesenchymal transition-related markers. We identified p53 as a regulator of LINP1, and LINP1 overexpression could restore the metastatic effects of p53. Furthermore, LINP1 was upregulated in doxorubicin- and 5-fluorouracil-resistant cells and induced chemoresistance. We also observed that LINP1 enrichment played a critical functional role in chemoresistance by inhibiting chemotherapeutics-induced apoptosis. Moreover, LINP1 in tumors was associated with lower overall survival and disease-free survival. In conclusion, LINP1 may serve as a potential oncogene and chemoresistance-related regulator of breast cancer cells, suggesting that LINP1 might be a potent therapeutic target and might reduce chemoresistance in breast cancer.

Macrophages in tumor microenvironment are mostly M2-polarized - and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). Here, we examined the regulatory effects of Huaier extract on TAMs using RAW264.7 murine macrophage cell line. Our data demonstrated that Huaier extract could inhibit the infiltration of macrophages into tumor microenvironment in a dose-dependent manner. By performing RT-PCR, immunofluorescence and phagocytosis assay, we were able to find that Huaier extract could regulate the polarization of macrophages, with decreased M2-polarization and increased phagocytosis of RAW264.7 cells. Moreover, we identified that Huaier extract could suppress macrophages-induced angiogenesis by using HUVEC migration assay, tube formation and chorioallantoic membrane assay. Additionally, western blotting showed decreased expression of MMP2, MMP9 and VEGF with the use of Huaier extract. Finally, we found that Huaier extract could inhibit M2-macrophages infiltration and angiogenesis through treating 4T1 tumor bearing mice with Huaier extract. Our study revealed a novel mechanism of the anti-tumor effect of Huaier extract which inhibited angiogenesis by targeting TAMs. These findings provided that Huaier was a promising drug for clinical treatment of breast cancer.