Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the correct folding and functionality of its client protein. Numerous Hsp90-client proteins are involved in cancer development. Thus, Hsp90 inhibitors have potential applications as anti-cancer drugs. We previously discovered that Hsp90α expression increased in breast cancer stem cells (BCSCs), which can initiate tumorigenesis and metastasis and resist treatment. In the present study, we further demonstrated that 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could suppress the self-renewal of BCSCs by downregulating B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a polycomb family member with oncogenic activity in breast cancer. Through immunoprecipitation analysis, we found that BMI1 did not interact with Hsp90α and that the downregulation of BMI1 by 17-DMAG was mediated by the inhibition of c-Myc and enhancement of zeste homolog 2 (EZH2) expression. The transcriptional and BMI1 promoter-binding activities of c-Myc in BCSCs were inhibited by 17-DMAG treatment. The overexpression of EZH2 attenuated the inhibitory effect of 17-DMAG on BMI1 and c-Myc expression. Furthermore, Hsp90α could be co-immunoprecipitated with c-Myc and EZH2 and bind to the BMI1 promoter. Treatment with 17-DMAG decreased the nuclear expression of EZH2 and c-Myc but not that of Hsp90α. In conclusion, our data suggested that Hsp90α could positively regulate the self-renewal of BCSCs by facilitating the nuclear translocation of c-Myc and EZH2 to maintain BMI1 expression.

Breast cancer is the most common cancer for women in Taiwan and post-lumpectomy radiotherapy is one of the therapeutic strategies for this malignancy. Although the 10-year overall survival of breast cancer patients is greatly improved by radiotherapy, the locoregional recurrence is around 10% and triple negative breast cancers (TNBCs) are at a high risk for relapse. The aim of this paper is to understand the mechanisms of radioresistance in breast cancers which may facilitate the development of new treatments in sensitizing breast cancer toward radiation therapy. Tribbles homolog 3 (TRIB3) is a pseudokinase protein and known to function as a protein scaffold within cells. It has been reported that higher TRIB3 expression is a poor prognostic factor in breast cancer patients with radiotherapy. In this study, we investigate the involvement of TRIB3 in the radiation response of TNBC cells. We first found that the expression of TRIB3 and the activation of Notch1, as well as Notch1 target genes, increased in two radioresistant TNBC cells. Knockdown of TRIB3 in radioresistant MDA-MB-231 TNBC cells decreased Notch1 activation, as well as the CD24-CD44+ cancer stem cell population, and sensitized cells toward radiation treatment. The inhibitory effects of TRIB3 knockdown in self-renewal or radioresistance could be reversed by forced expression of the Notch intracellular domain. We also observed an inhibition in cell growth and accumulated cells in the G0/G1 phase in radioresistant MDA-MB-231 cells after knockdown of TRIB3. With immunoprecipitation and mass spectrometry analysis, we found that, BCL2-associated transcription factor 1 (BCLAF1), BCL2 interacting protein 1 (BNIP1), or DEAD-box helicase 5 (DDX5) were the possible TRIB3 interacting proteins and immunoprecipitation data also confirmed that these proteins interacted with TRIB3 in radioresistant MDA-MB-231 cells. In conclusion, the expression of TRIB3 in radioresistant TNBC cells participated in Notch1 activation and targeted TRIB3 expression may be a strategy to sensitize TNBC cells toward radiation therapy.

Breast cancer brain metastasis commonly occurs in one-fourth of breast cancer patients and is associated with poor prognosis. Abnormal glucose metabolism is found to promote cancer metastasis. Moreover, the tumor microenvironment is crucial and plays an active role in the metabolic adaptations and survival of cancer cells. Glucose transporters are overexpressed in cancer cells to increase glucose uptake. The glucose transporter 3 (GLUT3) is a high-affinity glucose transporter that is highly expressed in mammalian neurons. GLUT3 is also overexpressed in several malignant brain tumors. However, the role of GLUT3 in breast cancer brain metastasis remains unknown. The results of the present study demonstrated that GLUT3 is highly overexpressed in brain metastatic breast cancers and mediates glucose metabolic reprogramming. Furthermore, knockdown of cAMP-response element binding protein (CREB) could directly regulate GLUT3 expression in brain metastatic breast cancer cells. Notably, we verified and provided a novel role of GLUT3 in mediating glucose metabolism and assisting breast cancer cells to survive in the brain to promote brain metastasis.

Radioresistant cells cause recurrence in patients with breast cancer after they undergo radiation therapy. The molecular mechanisms by which cancer cells obtain radioresistance should be understood to develop radiation-sensitizing agents. Results showed that the protein expression and activity of NAD(P)H:quinone oxidoreductase 1 (NQO1) were upregulated in radioresistant MDA-MB-231 triple-negative breast cancer (TNBC) cells. NQO1 knockdown inhibited the proliferation of NQO1 expressing Hs578t TNBC cells or the radioresistant MDA-MB-231 cells, whereas NOQ1 overexpression increased the survival of MDA-MB-231 cells, which lack of NQO1 expression originally, under irradiation. The cytotoxicity of β-lapachone, an NQO1-dependent bioactivatable compound, was greater in radioresistant MDA-MB-231 cells than in parental cells. β-lapachone displayed a radiosensitization effect on Hs578t or radioresistant MBDA-MB-231 cells. The expression of the long noncoding RNA NEAT1 positively regulated the NQO1 expression in radioresistant MDA-MB-231 cells at a translational level rather than at a transcription level. The inhibition of the NEAT1 expression through the CRISPR-Cas9 method increased the sensitivity of radioresistant MDA-MB-231 cells to radiation and decreased their proliferation, the activity of cancer stem cells, and the expression of stemness genes, including BMI1, Oct4, and Sox2. In conclusion, the NQO1 expression in triple-negative breast cancer cells determined their radiosensitivity and was controlled by NEAT1. In addition, NOQ1 bioactivatable compounds displayed potential for application in the development of radiation sensitizers in breast cancer.