CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner.

Great progress has been made in the field of tumor immunotherapy in the past decade. However, the therapeutic effects of immune checkpoint blockade (ICB) against ovarian cancer are still limited. Recently, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6i) has been reported to enhance antitumor immunity in preclinical models. The combined use of CDK4/6i and ICB may be beneficial, but the effects of CDK4/6is on the tumor immune microenvironment and whether they can synergize with ICB in treating ovarian cancer remain unknown. Methods: In this study, we first assessed the antitumor efficacy of abemaciclib, an FDA-approved CDK4/6i, in a syngeneic murine ovarian cancer model. Then, immunohistochemistry, immunofluorescence and flow cytometry were performed to evaluate the number, proportion, and activity of tumor-infiltrating lymphocytes. Cytokine and chemokine production was detected both in vivo and in vitro by PCR array analysis and cytokine antibody arrays. The treatment efficacy of combined abemaciclib and anti-PD-1 therapy was evaluated in vivo, and CD8+ and CD4+ T cell activities were analyzed using flow cytometry. Lastly, the requirement for both CD8+ T cells and B cells in combination treatment was evaluated in vivo, and potential cellular mechanisms were further analyzed by flow cytometry. Results: We observed that abemaciclib monotherapy could enhance immune infiltration, especially CD8+ T cell and B cell infiltration, in the ID8 murine ovarian cancer model. Immunophenotyping analysis showed that abemaciclib induced a proinflammatory immune response in the tumor microenvironment. PCR array analysis suggested the presence of a Th1-polarized cytokine profile in abemaciclib-treated ID8 tumors. In vitro studies showed that abemaciclib-treated ID8 cells secreted more CXCL10 and CXCL13, thus recruiting more lymphocytes than control groups. Combination treatment achieved better tumor control than monotherapy, and the activities of CD8+ and CD4+ T cells were further enhanced when compared with monotherapy. The synergistic antitumor effects of combined abemaciclib and anti-PD-1 therapy depended on both CD8+ T cells and B cells. Conclusion: These findings suggest that combined treatment with CDK4/6i and anti-PD-1 antibody could improve the efficacy of anti-PD-1 therapy and hold great promise for the treatment of poorly immune-infiltrated ovarian cancer.

Ermin is a p116RIP -interacting protein promoting oligodendroglial differentiation and myelin maintenance.

Myelin sheaths, which insulate the axons and ensure saltatory conduction of the nerve impulse, are generated and maintained via largely uncharacterized mechanisms. Ermin is an oligodendrocyte-specific protein associated with the cytoskeleton, but how it regulates cytoskeletal remodeling during oligodendrocyte differentiation and its role in myelin maintenance are not clear. To address this, we generated mice constitutively deficient for Ermn, the Ermin-coding gene. We found that aged Ermn-knockout mice exhibit an aberrant myelin architecture, with splitting of myelin layers, peeling of the myelin sheath from axons, and breakdown of myelinated fibers. As a result, these mice had remarkably impaired motor coordination. Ermn knockout also accelerated cuprizone-induced demyelination and exacerbated the associated movement disorders. Ermin was found to contribute to oligodendrocyte morphogenesis by associating with the myosin phosphatase Rho interacting protein (Mprip/p116RIP ) and inactivating RhoA, a GTPase that controls cytoskeletal rearrangement in differentiating cells. These findings provide novel insights into the mechanisms regulating oligodendroglial differentiation, the maintenance of the myelin sheaths, and remyelination.

Erratum: Tanshinone IIA and Astragaloside IV promote the angiogenesis of mesenchymal stem cell-derived endothelial cell-like cells via upregulation of Cx37, Cx40 and Cx43.

[This corrects the article DOI: 10.3892/etm.2017.5636.].

Retraction Note: Nuclear factor of activated T cells 5 maintained by Hotair suppression of miR-568 upregulates S100 calcium binding protein A4 to promote breast cancer metastasis.

The authors are retracting this article [1] after an investigation by the Ethics Committee of the Fourth Military Medical University (Xi'an, Shaanxi, China) of the following concerns that had been raised with respect to two of the figures.

Long non-coding RNAs AC026904.1 and UCA1: a "one-two punch" for TGF-ß-induced SNAI2 activation and epithelial-mesenchymal transition in breast cancer.

Transforming growth factor-ß (TGF-ß) has received much attention as a major inducer of epithelial-mesenchymal transition (EMT) during cancer progression, mainly by activating a set of pleiotropic transcription factors including SNAI2/Slug. However, the involvement of long non-coding RNAs (lncRNAs) in TGF-ß-induced Slug activation and EMT remains largely unknown. Methods: In this study, we used microarray analysis to compare lncRNA expression profiles between TGF-ß treated and untreated breast cancer cells. Then, the clinical significance of lncRNAs in breast cancer was investigated by qPCR and Kaplan-Meier survival analysis. The molecular mechanisms and EMT-promoting effects in vitro were analyzed by confocal laser microscopy, Western blotting, chromosome conformation capture (3C), chromatin isolation by RNA purification (ChIRP), ChIP, luciferase reporter assay and transwell migration assay. Lastly, the pro-metastatic effects in vivo were evaluated by bioluminescent imaging and hematoxylin and eosin (H&E) staining. Results: We observed that TGF-ß induced genome-wide changes in lncRNA levels in breast cancer cells, among which AC026904.1 and UCA1 were highly expressed in metastatic breast cancer and closely associated with poor prognosis. Mechanistic study revealed that AC026904.1 and UCA1 were upregulated by non-canonical and canonical TGF-ß pathways, respectively. Further analysis showed that AC026904.1 functions as an enhancer RNA in the nucleus, whereas UCA1 exerts a competitive endogenous RNA (ceRNA) activity in the cytoplasm. In addition, the biological functions of these two lncRNAs converged on the activation and maintenance of Slug, constituting a one-two punch in promoting EMT and tumor metastasis. Conclusion: These findings uncover for the first time that AC026904.1 and UCA1 could cooperatively upregulate Slug expression at both transcriptional and post-transcriptional levels, exerting critical roles in TGF-ß-induced EMT. The present work provides new evidence that lncRNAs function as key regulators of EMT and hold great promise to be used as novel biomarkers and therapeutic targets for metastatic breast cancer.

Tanshinone IIA and Astragaloside IV promote the angiogenesis of mesenchymal stem cell-derived endothelial cell-like cells via upregulation of Cx37, Cx40 and Cx43.

Tanshinone IIA (Tan IIA) and Astragaloside IV (AGS-IV) were used as therapeutic treatments for coronary heart diseases (CHDs) in ancient China. However, the underlying mechanisms mediating the effects of Tan IIA and AGS-IV in angiogenesis remain unknown. In the present study, mesenchymal stem cells (MSCs) were induced to differentiate into endothelial cell (EC)-like cells in vitro and the effects of Tan IIA and/or AGS-IV on the functions of these cells, including cell proliferation and tube formation, were assessed. Compared with the single-agent groups (Tan IIA or AGS-IV only), combined-agent (Tan IIA and AGS-IV) treatment significantly enhanced the proliferation and tube formation capacity of EC-like cells. In addition, the expression of connexin 37 (Cx37), Cx40 and Cx43 in the combined-agent group was significantly increased compared with the single-agent groups. Furthermore, enhanced gap junctional intercellular communication (GJIC) was identified in the combined-agent group, as evidenced by increased dye transfer in scrape-loading dye transfer assays. In conclusion, Tan IIA and AGS-IV may promote the angiogenesis of EC-like cells by upregulating the expression of Cx37, Cx40 and Cx43 and enhancing GJIC function. The results of the present study may provide experimental evidence for the clinical application of Tan IIA and AGS-IV as a treatment for CHDs.

Long non-coding RNA UCA1 desensitizes breast cancer cells to trastuzumab by impeding miR-18a repression of Yes-associated protein 1.

Breast cancer resistance to the monoclonal erbB2/HER2 antibody trastuzumab (or herceptin) has become a significant obstacle in clinical targeted therapy of HER2-positive breast cancer. Previous research demonstrated that such drug resistance may be related to dysregulation of miRNA expression. Here, we found that knockdown of the long non-coding RNA, urothelial cancer associated 1 (UCA1), can promote the sensitivity of human breast cancer cells to trastuzumab. Mechanistically, UCA1 knockdown upregulated miR-18a and promoted miR-18a repression of Yes-associated protein 1 (YAP1). A luciferase reporter assay confirmed the association of miR-18a with wild-type UCA1 but not with UCA1 mutated at the predicted miR-18a-binding site. The direct targeting of YAP1 by miR-18a was verified by the observation that miR-18a mimic suppressed luciferase expression from a construct containing the YAP1 3' untranslated region. Meanwhile, reciprocal repression of UCA1 and miR-18a were found to be Argonaute 2-dependent. Knockdown of YAP1 recapitulated the effect of UCA1 silencing by reducing the viability of trastuzumab-treated breast cancer cells, whereas inhibition of miR-18a abrogated UCA1 knockdown-induced improvement of trastuzumab sensitivity in breast cancer cells. These findings demonstrate that the UCA1/miR-18a/YAP1 axis plays an important role in regulating the sensitivity of breast cancer cells to trastuzumab, which has implications for the development of novel approaches to improving breast cancer responses to targeted therapy.

Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor.

Chimeric antigen receptor-modulated T lymphocytes (CAR-T) have emerged as a powerful tool for arousing anticancer immunity. Endogenous ligands for tumor antigen may outperform single-chain variable fragments to serve as a component of CARs with high cancer recognition efficacy and minimized immunogenicity. As heterodimerization and signaling partners for human epidermal growth factor receptor 2 (HER2), HER3/HER4 has been implicated in tumorigenic signaling and therapeutic resistance of breast cancer. In this study, we engineered T cells with a CAR consisting of the extracellular domain of heregulin-1ß (HRG1ß) that is a natural ligand for HER3/HER4, and evaluated the specific cytotoxicity of these CAR-T cells in cultured HER3 positive breast cancer cells and xenograft tumors. Our results showed that HRG1ß-CAR was successfully constructed, and T cells were transduced at a rate of 50%. The CAR-T cells specifically recognized and killed HER3-overexpressing breast cancer cells SK-BR-3 and BT-474 in vitro, and displayed potent tumoricidal effect on SK-BR-3 xenograft tumor models. Our results suggest that HRG1ß-based CAR-T cells effectively suppress breast cancer driven by HER family receptors, and may provide a novel strategy to overcome cancer resistance to HER2-targeted therapy.

Missing Links in Epithelial-Mesenchymal Transition: Long Non-Coding RNAs Enter the Arena.

Cancer metastasis occurs through a series of sequential steps, which involves dissemination of tumor cells from a primary site and colonization in distant tissues. To promote the invasion-metastasis cascade, carcinoma cells usually initiate a cell-biological program called epithelial-mesenchymal transition (EMT), which is orchestrated by a set of master regulators, including TGF-ß, Snail, ZEB and Twist families. The biological activities of these molecules are tightly regulated by a variety of cell-intrinsic pathways as well as extracellular cues. Recently, accumulating evidence indicates that long non-coding RNAs (lncRNAs) represent some of the most differentially expressed transcripts between primary and metastatic cancers. LncRNAs including MALAT1, HOTAIR, H19, LncRNA-ATB, and LincRNA-ROR have been reported to be involved in the process of EMT, mainly through cross-talking with master regulators of EMT. Thus, understanding the different and precise molecular mechanisms by which functional lncRNAs switch EMT on and off is important for opening up new avenues in lncRNA-directed diagnosis, prognosis, and therapeutic intervention against cancer.

Targeted inhibition of hantavirus replication and intracranial pathogenesis by a chimeric protein-delivered siRNA.

Hantavirus (HV) infection, which underlies hantavirus hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, remains to be a severe clinical challenge. Here, we synthesized small interfering RNAs (siRNAs) that target the encoding sequences of HV strain 76-118, and validated their inhibitory role in virus replication in HV-infected monkey kidney Vero E6 cells. A chimeric protein, 3G1-Cκ-tP, consisting of a single-chain antibody fragment (3G1) against the HV surface envelop glycoprotein, the constant region of human immunoglobulin κ chain (Cκ), and truncated protamine (amino acids 8-29, tP), was further generated. The fusion protein showed high affinity to HV antigen on the infected cell membrane, and internalized through clathrin-mediated endocytosis; it bound to siRNAs via the basic nucleic acid-rich protamine fragment, leading to their specific delivery into HV-infected cells and efficient inhibition of virus replication. An encephalitis mouse model was established via intracranial HV administration. Intraperitoneal injection of siRNAs complexed with 3G1-Cκ-tP achieved specific distribution of siRNAs in HV-infected brain cells, significantly reduced HV antigen levels, and effective protection from HV infection-derived animal death. These results provide a compelling rationale for novel therapeutic protocols designed for HV infection and related disorders.

Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling.

Neutrophil release of NO/ONOO- induces endothelial cell barrier dysfunction in inflammatory acute lung injury (ALI). Previous studies using zymosan-triggered inflammation and ALI model revealed that zymosan promotes inducible NO synthase (iNOS) expression in neutrophils, and that isoflurane inhibits zymosan-induced oxidative stress and iNOS biosynthesis. However, the underlying mechanisms remain largely unknown. We found here that in zymosan-primed neutrophils, iNOS is transcriptionally activated by NF-κB, whose nuclear translocation is triggered by excessive reactive oxygen species (ROS) and consequently activated p38 MAPK. ROS production is attributed to zymosan-initiated Toll-like receptor 2 (TLR2) signaling, in which the adaptor MyD88 recruits and activates c-Src, and c-Src activates NADPH oxidase to generate ROS. Subanesthetic isoflurane counteracts the aforementioned zymosan-induced signaling by targeting N-methyl-D-aspartic acid (NMDA) glutamate receptor and thereby suppressing calcium influx and c-Src activation. Whereas iNOS accelerates NO/ONOO- production in neutrophils which eventually promote protein leak from pulmonary microvascular endothelial cells (PMVEC), isoflurane reduced NO/ONOO- release from zymosan-treated neutrophils, and thus relieves trans-PMVEC protein leak. This study provides novel insights into the roles of neutrophils and the underlying mechanisms in zymosan-induced ALI, and has implications for the therapeutic potential of subanesthetic isoflurane in attenuating inflammatory responses causing lung endothelial cell damage.

Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma.

Ubiquitin-specific protease 22 (USP22) aberrance has been implicated in several malignancies; however, whether USP22 plays a role in anaplastic thyroid carcinoma (ATC) remains unclear. Here, we report that USP22 expression is highly elevated in ATC tissues, which positively correlated with tumor size, extracapsular invasion, clinical stages, and poor prognosis of ATC patients. In vitro assays showed that USP22 depletion suppressed ATC cell survival and proliferation by decreasing Rb phosphorylation and cyclin D2, inactivating Akt, and simultaneously upregulating Rb; USP22 silencing restrained cell migration and invasion by inhibiting epithelial-mesenchymal transition; USP22 knockdown promoted mitochondrion- mediated and caspase-dependent apoptosis by upregulating Bax and Bid and promoting caspase-3 activation. Consistent with in vitro findings, downregulation of USP22 in ATC cells impeded tumor growth and lung metastasis in vivo. These results raise the applicability for USP22 as a useful predictor of ATC prognosis and a potential therapeutic target for ATC.

MiRNA-101 inhibits breast cancer growth and metastasis by targeting CX chemokine receptor 7.

Whereas miR-101 is involved in the development and progression of breast cancer, the underlying molecular mechanisms remain to be elucidated. Here, we report that miR-101 expression is inversely correlated with the clinical stage, lymph node metastasis and prognosis in breast cancers. Introduction of miR-101 inhibited breast cancer cell proliferation and invasion in vitro and suppressed tumor growth and lung metastasis of in vivo. CX chemokine receptor 7 (CXCR7) is a direct target of miR-101, positively correlating with the histological grade and the incidence of lymph node metastasis in breast cancer patients. The effects of miR-101 were mimicked and counteracted by CXCR7 depletion and overexpression, respectively. STAT3 signaling downstream of CXCR7 is involved in miR-101 regulation of breast cancer cell behaviors. These findings have implications for the potential application of miR-101 in breast cancer treatment.

MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha.

The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3'-untranslated region (3'UTR) of TOP2a mRNA. Further more, we revealed that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment.

Regulators of carcinogenesis: emerging roles beyond their primary functions.

Cancers are characterized by aberrant cell signaling that results in accelerated proliferation, suppressed cell death, and reprogrammed metabolism to provide sufficient energy and intermediate metabolites for macromolecular biosynthesis. Here, we summarize the emerging "unconventional" roles of these regulators based on their newly identified interaction partners, different subcellular localizations, and/or structural variants. For example, the epidermal growth factor receptor (EGFR) regulates DNA synthesis, microRNA maturation and drug resistance by interacting with previously undescribed partners; cyclins and cyclin-dependent kinases (CDKs) crosstalk with multiple canonical pathways by phosphorylating novel substrates or by functioning as transcriptional factors; apoptosis executioners play extensive roles in necroptosis, autophagy, and in the self-renewal of stem cells; and various metabolic enzymes and their mutants control carcinogenesis independently of their enzymatic activity. These recent findings will supplement the systemic functional annotation of cancer regulators and provide new rationales for potential molecular targeted cancer treatments.

MicroRNA-26a promotes anoikis in human hepatocellular carcinoma cells by targeting alpha5 integrin.

Metastasis is the major reason for the death of patients suffering from malignant diseases such as human hepatocellular carcinoma (HCC). Among the complex metastatic process, resistance to anoikis is one of the most important steps. Previous studies demonstrate that microRNA-26a (miR-26a) is an important tumor suppressor that inhibits the proliferation and invasion of HCC cells by targeting multiple oncogenic proteins. However, whether miR-26a can also influence anoikis has not been well established. Here, we discovered that miR-26a promotes anoikis of HCC cells both in vitro and in vivo. With a combinational analysis of bioinformatics and public clinical databases, we predicted that alpha5 integrin (ITGA5), an integrin family member, is a putative target of miR-26a. Furthermore, we provide experimental evidence to confirm that ITGA5 is a bona fide target of miR-26a. Through gain- and loss-of-function studies, we demonstrate that ITGA5 is a functional target of miR-26a-induced anoikis in HCC cells. Collectively, our findings reveal that miR-26a is a novel player during anoikis and a potential therapeutic target for the treatment of metastatic HCC.

Post-transcriptional up-regulation of PDGF-C by HuR in advanced and stressed breast cancer.

Breast cancer is a heterogeneous disease characterized by multiple genetic alterations leading to the activation of growth factor signaling pathways that promote cell proliferation. Platelet-derived growth factor-C (PDGF-C) is overexpressed in various malignancies; however, the involvement of PDGF-C in breast cancers and the mechanisms underlying PDGF-C deregulation remain unclear. Here, we show that PDGF-C is overexpressed in clinical breast cancers and correlates with poor prognosis. PDGF-C up-regulation was mediated by the human embryonic lethal abnormal vision-like protein HuR, which stabilizes the PDGF-C transcript by binding to two predicted AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). HuR is up-regulated in hydrogen peroxide-treated or ultraviolet-irradiated breast cancer cells. Clinically, HuR levels are correlated with PDGF-C expression and histological grade or pathological tumor-node-metastasis (pTNM) stage. Our findings reveal a novel mechanism underlying HuR-mediated breast cancer progression, and suggest that HuR and PDGF-C are potential molecular candidates for targeted therapy of breast cancers.

Nuclear factor of activated T cells 5 maintained by Hotair suppression of miR-568 upregulates S100 calcium binding protein A4 to promote breast cancer metastasis.

INTRODUCTION: The onset of distal metastasis, which underlies the high mortality of breast cancers, warrants substantial studies to depict its molecular basis. Nuclear factor of activated T cells 5 (NFAT5) is upregulated in various malignancies and is critically involved in migration and invasion of neoplastic cells. Nevertheless, the metastasis-related events potentiated by this transcriptional factor and the mechanism responsible for NFAT5 elevation in carcinoma cells remain to be fully elucidated. METHODS: The correlation of NFAT5 with breast cancer invasiveness was investigated in vitro and clinically. The genes transcriptionally activated by NFAT5 were probed and their roles in breast cancer progression were dissected. The upstream regulators of NFAT5 were studied with particular attempt to explore the involvement of non-coding RNAs, and the mechanism underlying the maintenance of NFAT5 expression was deciphered. RESULTS: In metastatic breast cancers, NFAT5 promotes epithelial-mesenchymal transition (EMT) and invasion of cells by switching on the expression of the calcium binding protein S100A4, and facilitates the angiogenesis of breast epithelial cells and thus the development of metastases by transcriptionally activating vascular endothelial growth factor C (VEGF-C). NFAT5 is directly targeted by miR-568, which is in turn suppressed by the long non-coding RNA, Hotair, via a documented in trans gene silencing pattern, that is recruitment of the polycomb complex (Polycomb Repressive Complex 2; PRC2) and LSD1, and consequently methylation of histone H3K27 and demethylation of H3K4 on the miR-568 loci. CONCLUSION: This study unravels a detailed role of NFAT5 in mediating metastatic signaling, and provides broad insights into the involvement of Hotair, in particular, by transcriptionally regulating the expression of microRNA(s), in the metastasis of breast cancers.

MiR-200c suppresses TGF-ß signaling and counteracts trastuzumab resistance and metastasis by targeting ZNF217 and ZEB1 in breast cancer.

Resistance to trastuzumab and concomitantly distal metastasis are leading causes of mortality in HER2-positive breast cancers, the molecular basis of which remains largely unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased tumorigenicity and invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-ß signaling in these cells. MiR-200c, which was the most significantly downregulated miRNA in trastuzumab-resistant cells, restored trastuzumab sensitivity and suppressed invasion of breast cancer cells by concurrently targeting ZNF217, a transcriptional activator of TGF-ß, and ZEB1, a known mediator of TGF-ß signaling. Given the reported backward inhibition of miR-200c by ZEB1, ZNF217 also exerts a feedback suppression of miR-200c via TGF-ß/ZEB1 signaling. Restoration of miR-200c, silencing of ZEB1 or ZNF217 or blockade of TGF-ß signaling increased trastuzumab sensitivity and suppressed invasiveness of breast cancer cells. Therefore, our study unraveled nested regulatory circuits of miR-200c/ZEB1 and miR-200c/ZNF217/TGF-ß/ZEB1 in synergistically promoting trastuzumab resistance and metastasis of breast cancer cells. These findings provide novel insights into the common role of EMT and related molecular machinery in mediating the malignant phenotypes of breast cancers.

Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.

BACKGROUND: Resistance to humanized monoclonal erbB2/HER2 antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers. The activation of alternative growth factor receptors, in particular, the insulin-like growth factor 1 receptor (IGF1R), represents a common feature of trastuzumab-refractory cells; however, the underlying mechanism remains elusive. METHODS: Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3 cells in the presence of trastuzumab. Among the differentially expressed microRNAs (miRNAs) screened by microarray analysis, candidate miRNA(s) predicted to target IGF1R was studied for its role in conferring trastuzumab resistance. The mechanism underlying decreased expression of IGF1R-targeted miRNA in refractory cells was also addressed. RESULTS: miR-375, which was downregulated and predicted to target IGF1R in trastuzumab-resistant HER2-positive breast cancer cells, could indeed inhibit the cellular luciferase activity in a reporter construct containing the 3'-UTR of IGF1R. Overexpression of miR-375 restored the sensitivity of cells to trastuzumab, while inhibition of miR-375 conferred trastuzumab resistance on HER2-positive breast cancer cells. Blockade of DNA methylation and histone deacetylation restored the expression of miR-375 in trastuzumab-resistant cells. A reverse correlation between the levels of miR-375 and IGF1R was validated in clinical breast cancers. CONCLUSIONS: Epigenetic silencing of miR-375 causes the upregulation of IGF1R, which at least partially underlies trastuzumab resistance of breast cancer cells. Our study has implications for miR-375 as a potential target in combination with trastuzumab for treating HER2-positive breast cancers.