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The dysregulation of miRNAs has been increasingly recognized as a critical mediator of cancer development and progression. Here, we show that frequent deletion of the MIR135A1 locus is associated with poor prognosis in primary breast cancer. Forced expression of miR-135a decreased breast cancer progression, while inhibition of miR-135a with a specific miRNA sponge elicited opposing effects, suggestive of a tumor suppressive role of miR-135a in breast cancer. Estrogen receptor alpha (ERα) bound the promoter of MIR135A1 for its transcriptional activation, whereas tamoxifen treatment inhibited expression of miR-135a in ERα+ breast cancer cells. miR-135a directly targeted ESR1, ESRRA, and NCOA1, forming a negative feedback loop to inhibit ERα signaling. This regulatory feedback between miR-135a and ERα demonstrated that miR-135a regulated the response to tamoxifen. The tamoxifen-mediated decrease in miR-135a expression increased the expression of miR-135a targets to reduce tamoxifen sensitivity. Consistently, miR-135a expression was downregulated in ERα+ breast cancer cells with acquired tamoxifen resistance, while forced expression of miR-135a partially resensitized these cells to tamoxifen. Tamoxifen resistance mediated by the loss of miR-135a was shown to be partially dependent on the activation of the ERK1/2 and AKT pathways by miR-135a–targeted genes. Taken together, these results indicate that deletion of the MIR135A1 locus and decreased miR-135a expression promote ERα+ breast cancer progression and tamoxifen resistance.Significance: Loss of miR-135a in breast cancer disrupts an estrogen receptor-induced negative feedback loop, perpetuating disease progression and resistance to therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4915/F1.large.jpg. Cancer Res; 78(17); 4915–28. ©2018 AACR.

Supplementary table S1 lists all the sequence of miR-135a mimic, siRNAs, shRNAs, and primers for plasmid construction, ChIP assay, and qRT-PCR used in this study. Supplementary table S2 shows the information of antibodies used in this study. Supplementary table S3 and S4 show the correlation of MIR135A1 deletion and low miR-135a-1 with the clinicopathologic features of breast cancer in the TCGA breast tumors, respectively. Supplementary table S5 lists genes including EMT markers, ERα cofactors and interacting proteins, and predicted miR-135a targets which expression levels negatively correlate with miR-135a in breast tumors. Supplementary figure S1 shows that the copy number of MIR135A1 gene and the expression levels of pri-miR-135a-1 are decreased in breast cancer. Supplementary figure S2 shows that miR-135a is a tumor suppressor gene in T47D and MDA-MB-231 cells and inhibits EMT of breast cancer cells. Supplementary figure S3 shows that miR-135a impairs the in vivo tumor growth. Supplementary figure S4 shows that the transcription of MIR135A1 positively correlates with E2-ERα axis. Supplementary figure S5 shows that miR-135a modulates the estrogen independent growth and the response to tamoxifen in ERα+ breast cancer cells. Supplementary figure S6 shows the validation of miR-135a targets in breast cancer cells. Supplementary figure S7 shows that miR-135a modulates ERα-ERK-AKT crosstalk.

PDF file - 1291K, FIGURE S1: Representative data of genomic real-time PCR screening for DNA copy numbers of aberrant microRNAs on human chromosome 3p in breast carcinoma and the correlation between expression levels of miR-128 and ARPP21; FIGURE S2: miR-128 depletion confers non-tumorigenic MCF-10A cells with a malignant phenotype in vitro; FIGURE S3: Forced expression of miR-128 alters the morphology and reduces migration and invasion of MCF-7 cells in vitro, whereas miR-128 depletion confers non-tumorigenic HBL-100 cells with a malignant phenotype in vitro; FIGURE S4: miR-128 depletion confers a malignant phenotype on non-tumorigenic MCF-10A cells in vivo and miR-128 represses the CD44+CD24 Neg/Low population in mammary carcinoma cells; FIGURE S5: Interactions between miR-128 and its binding targets; FIGURE S6: Luciferase assay for promoter activity

PDF file - 226K, TABLE S1: List of miRNAs on human chromosome 3p and the changes of their genome copies determined by genomic real-time PCR; TABLE S2: Clinical significance of the expression of miR-128 in breast cancer patients; TABLE S3: Invariable analysis of clinical outcome according to the expression of miR-128 in breast cancer patients (85 cases); TABLE S4: List of mRNAs predicted to be targeted by miR-128 by all the three programs: MiRanda, Pictar and TargetScan; TABLE S5: qRT-PCR analysis of the effect of forced expression of miR-128 in MDA-MB-231 cells or depleted expression of miR-128 in MCF-10A cells on mRNA levels of several key genes functionally involved in oncogenicity and neoplastic progression; TABLE S6: Sequences of the oligonucleotides for miRNAs, siRNAs, construct-making and ChIP assays; TABLE S7: Sequence of the oligonucleotides for real-time PCR experiments; TABLE S8: List of proteins tested by antibodies and characteristics of the corresponding antibodies used

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Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer. Cancer Res; 71(20); 6463–74. ©2011 AACR.

PDF file - 190K, Includes patient and specimen data, reagent information, miRNA and siRNA transfection, and statistical analysis. Also includes supplementary references

The dysregulation of miRNAs has been increasingly recognized as a critical mediator of cancer development and progression. Here, we show that frequent deletion of the MIR135A1 locus is associated with poor prognosis in primary breast cancer. Forced expression of miR-135a decreased breast cancer progression, while inhibition of miR-135a with a specific miRNA sponge elicited opposing effects, suggestive of a tumor suppressive role of miR-135a in breast cancer. Estrogen receptor alpha (ERα) bound the promoter of MIR135A1 for its transcriptional activation, whereas tamoxifen treatment inhibited expression of miR-135a in ERα+ breast cancer cells. miR-135a directly targeted ESR1, ESRRA, and NCOA1, forming a negative feedback loop to inhibit ERα signaling. This regulatory feedback between miR-135a and ERα demonstrated that miR-135a regulated the response to tamoxifen. The tamoxifen-mediated decrease in miR-135a expression increased the expression of miR-135a targets to reduce tamoxifen sensitivity. Consistently, miR-135a expression was downregulated in ERα+ breast cancer cells with acquired tamoxifen resistance, while forced expression of miR-135a partially resensitized these cells to tamoxifen. Tamoxifen resistance mediated by the loss of miR-135a was shown to be partially dependent on the activation of the ERK1/2 and AKT pathways by miR-135a–targeted genes. Taken together, these results indicate that deletion of the MIR135A1 locus and decreased miR-135a expression promote ERα+ breast cancer progression and tamoxifen resistance. Significance: Loss of miR-135a in breast cancer disrupts an estrogen receptor-induced negative feedback loop, perpetuating disease progression and resistance to therapy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4915/F1.large.jpg. Cancer Res; 78(17); 4915–28. ©2018 AACR.

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer. Cancer Res; 71(20); 6463–74. ©2011 AACR.