HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503

Mecanismes de la malaltia; Càncer de mamaMecanismos de la enfermedad; Cáncer de mamaDisease Mechanisms; Breast CancerThe HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3′UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222– MYB family–TIMP2 axis in regulation of motility in breast cancer cells.This work was supported by the Danish Council for Independent Research (grants no. 12-126942 and 12-127290 to SFP), by the Hartmann foundation (SFP), Fondation Juchum (SFP), Kirsten og Freddy Johansens Fond (SFP), the Breast Cancer Research Foundation (BCRF-17-008) (JA), Instituto de Salud Carlos III (PI16/00253) (JA) and the Harboe foundation (SFP). Katrine Franklin Mark is gratefully acknowledged for excellent technical assistance. We are grateful to Pascal Pineau from Institut Pasteur, France for the MYBL1 3′UTR/psiCHECK2 construct

<i>HER2</i>-encoded mir-4728 forms a receptor-independent circuit with miR-21-5p through the non-canonical poly(A) polymerase PAPD5

We previously reported that the human HER2 gene encodes the intronic microRNA mir-4728, which is overexpressed together with its oncogenic host gene and may act independently of the HER2 receptor. More recently, we also reported that the oncogenic miR-21-5p is regulated by 3′ tailing and trimming by the non-canonical poly(A) polymerase PAPD5 and the ribonuclease PARN. Here we demonstrate a dual function for the HER2 locus in upregulation of miR-21-5p; while HER2 signalling activates transcription of mir-21, miR-4728-3p specifically stabilises miR-21-5p through inhibition of PAPD5. Our results establish a new and unexpected oncogenic role for the HER2 locus that is not currently being targeted by any anti-HER2 therapy

MicroRNAs in HER2-Amplified Breast Cancer

Background: Breast cancer is the most common female malignancy and the leading cause of cancer-related deaths worldwide. Targeted therapy against the main biomarkers estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2/ERBB2) have greatly improved mortality rates, but ab initio or acquired therapy resistance is common. It is imperative to identify patients who will benefit from targeted therapy and develop new treatment modalities for resistant tumors. microRNAs (miRNAs) are small, non-coding RNAs that regulate expression of up to 60% of human protein-coding genes. Their frequent deregulation in cancer makes miRNAs attractive candidate biomarkers and clinical targets. We previously reported that the HER2 locus, which is amplified in 15-20% of breast cancers, also encodes an intronic miRNA, mir-4728. Aims: The aim of this thesis was to study the function of the main mature miRNA product of mir-4728, miR-4728-3p, and to investigate if this miRNA could serve as an independent diagnostic or treatment predictive biomarker in breast cancer. Results: Expression of miR-4728-3p correlated with HER2, and ER was identified as a direct target of the miRNA, connecting these two important breast cancer biomarkers. Regulation of ER occurred in a non-canonical manner, expanding our understanding of miRNA-mediated target gene regulation. During functional studies in vitro, we sometimes detected strong, batch-dependent off-target effects of commercially available miRNA mimics, stressing the need to maintain high quality standards in clinical studies using miRNA replacement therapy. Furthermore, inhibition of miR-4728-3p led to upregulation of the predicted target PAPD5, a non-canonical poly(A) polymerase, that destabilized the oncogenic microRNA miR-21. HER2 signaling is known to activate mir-21 transcription through the MAPK/ERK pathway, suggesting that the HER2 locus has a dual role in increasing levels of miR-21 through both miR-4728-3p and HER2. Blocking miR-4728-3p also strongly inhibited cellular metabolism, indicating a role for the miRNA in improved nutrient processing. Taken together, these results establish the importance of miR-4728-3p as a breast cancer oncogene

Passenger strand loading in overexpression experiments using microRNA mimics.

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand

The HER2-encoded miR-4728-3p regulates ESR1 through a non-canonical internal seed interaction.

Since the early 1980s remarkable progress has been made in understanding the role of the HER2 locus in carcinogenesis, but many details of its regulatory network are still elusive. We recently reported the finding of 367 new human microRNA (miRNA) genes of which one, mir-4728, is encoded in an intron of the HER2 gene. Here, we confirm that the HER2 oncogene is a bi-functional locus encoding the membrane receptor and a functional miRNA gene. We further show that miR-4728-3p has alternative functionalities depending on the region used for interaction with its target; the canonical seed between nucleotides 2-8 or a novel, more internal seed shifted to nucleotides 6-12. Analysis of public data shows that this internal seed region, although rare compared to the far more abundant canonical 2-8 seed interaction, can also direct targeted down-regulation by other miRNAs. Through the internal seed, miR-4728-3p regulates expression of estrogen receptor alpha, an interaction that would have remained undetected if classic rules for miRNA-target interaction had been applied. In summary, we present here an alternative mode of miRNA regulation and demonstrate this dual function of the HER2 locus, linking the two major biomarkers in breast cancer

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503

The HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3′UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222– MYB family–TIMP2 axis in regulation of motility in breast cancer cells

IS targeting is not restricted to miR-4728-3p.

<p>SylArray enrichment landscape from a microarray experiment of miR-30a overexpression in HCT116 cells. Data generated by Baraniskin <i>et al.</i> was analyzed by SylArray. Word designations and graph details as in Fig. 1a, with CS and IS referring to corresponding miR-30a positions.</p

IGV view (Integrative Genomics Viewer) showing the enrichment of AGO-CLIP sequencing reads in miR-4728-3p transfected vs non-transfected control around the IS target site on the 3′UTR of the Ubiquitin carboxyl-terminal hydrolase 1 (USP1) gene.

<p>The positions of the 7- and 6-mer IS target sites are highlighted below the USP1 gene.</p

miR-4728-3p IS regulates ESR1.

<p><b>A.</b> qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. <b>B.</b> Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an antisense oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). <b>C.</b> Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. <b>D.</b> MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). <b>E.</b> Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. <b>F.</b> Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of <0.05 (*), and <0.005 (**) in Student′s t-test.</p