Publications

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level.

Leukemic stem cells (LSCs) drive progression of chronic myeloid leukemia (CML) and tyrosine kinase inhibitor resistance through poorly understood mechanisms. Now in Cell Stem Cell, Zipeto et al. (2016) show targeting the RNA editing enzyme ADAR1 restores expression of let-7 and efficiently kills LSCs, providing an innovative therapeutic target in CML.

MicroRNAs (miRNA) are short, noncoding RNAs whose dysregulation has been implicated in most, if not all, cancers. They regulate gene expression by suppressing mRNA translation and reducing mRNA stability. To this end, there is a great deal of interest in modifying miRNA expression levels for the treatment of cancer. However, the literature is fraught with inconsistent accounts as to whether various miRNAs are oncogenic or tumor suppressive. In this review, we directly examine these inconsistencies and propose several mechanisms to explain them. These mechanisms include the possibility that specific miRNAs can simultaneously produce competing oncogenic and tumor suppressive effects by suppressing both tumor suppressive mRNAs and oncogenic mRNAs, respectively. In addition, miRNAs can modulate tumor-modifying extrinsic factors, such as cancer-immune system interactions, stromal cell interactions, oncoviruses, and sensitivity to therapy. Ultimately, it is the balance between these processes that determines whether a specific miRNA produces a net oncogenic or net tumor suppressive effect. A solid understanding of this phenomenon will likely prove valuable in evaluating miRNA targets for cancer therapy. Cancer Res; 76(13); 3666-70. ©2016 AACR.

In C. elegans, miRNAs are genetic biomarkers of aging. Similarly, multiple miRNAs are differentially expressed between younger and older persons, suggesting that miRNA-regulated biological mechanisms affecting aging are evolutionarily conserved. Previous human studies have not considered participants' lifespans, a key factor in identifying biomarkers of aging. Using PCR arrays, we measured miRNA levels from serum samples obtained longitudinally at ages 50, 55, and 60 from 16 non-Hispanic males who had documented lifespans from 58 to 92. Numerous miRNAs showed significant changes in expression levels. At age 50, 24 miRNAs were significantly upregulated, and 73 were significantly downregulated in the long-lived subgroup (76-92 years) as compared with the short-lived subgroup (58-75 years). In long-lived participants, the most upregulated was miR-373-5p, while the most downregulated was miR-15b-5p. Longitudinally, significant Pearson correlations were observed between lifespan and expression of nine miRNAs (p value<0.05). Six of these nine miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, 1225-3p) were also significantly up- or downregulated when comparing long-lived and short-lived participants. Twenty-four validated targets of these miRNAs encoded aging-associated proteins, including PARP1, IGF1R, and IGF2R. We propose that the expression profiles of the six miRNAs (miR-211-5p, 374a-5p, 340-3p, 376c-3p, 5095, and 1225-3p) may be useful biomarkers of aging.

SMARCA4 is the catalytic subunit of the SWI/SNF chromatin-remodeling complex, which alters the interactions between DNA and histones and modifies the availability of the DNA for transcription. The latest deep sequencing of tumor genomes has reinforced the important and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer. However, although SWI/SNF complex plays a key role in gene expression, the regulation of this complex itself is poorly understood. Significantly, an understanding of the regulation of SMARCA4 expression has gained in importance due to recent proposals incorporating it in therapeutic strategies that use synthetic lethal interactions between SMARCA4-MAX and SMARCA4-SMARCA2. In this report, we found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3'UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functional relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the aforementioned synthetic lethal therapeutic strategies.

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Lung cancer represents the leading cause of cancer-related deaths in men and women worldwide. Targeted therapeutics, including the epidermal growth factor receptor (EGFR) inhibitor erlotinib, have recently emerged as clinical alternatives for the treatment of non-small cell lung cancer (NSCLC). However, the development of therapeutic resistance is a major challenge, resulting in low 5-year survival rates. Due to their ability to act as tumor suppressors, microRNAs (miRNAs) are attractive candidates as adjuvant therapeutics for the treatment of NSCLC. In this study, we examine the ability of 2 tumor suppressor miRNAs, let-7b and miR-34a to sensitize KRAS;TP53 mutant non-small cell lung cancer cells to the action of erlotinib. Treatment with these miRNAs, individually or in combination, resulted in synergistic potentiation of the anti-proliferative effects of erlotinib. This effect was observed over a wide range of miRNA and erlotinib interactions, suggesting that let-7b and miR-34a target oncogenic pathways beyond those inhibited by EGFR. Combinatorial treatment with let-7b and miR-34a resulted in the strongest synergy with erlotinib, indicating that these miRNAs can effectively target multiple cellular pathways involved in cancer cell proliferation and resistance to erlotinib. Together, our findings indicate that NSCLC cells can be effectively sensitized to erlotinib by supplementation with tumor suppressor miRNAs, and suggest that the use of combinations of miRNAs as adjuvant therapeutics for the treatment of lung cancer is a viable clinical strategy.

The evolutionarily conserved gene lin-28 encodes an RNA-binding protein and is an important regulator of the proper temporal succession of several developmental events in both invertebrates and vertebrates. At the cellular level, LIN-28 promotes stemness and proliferation, and inhibits differentiation, a feature best illustrated by its ability to induce pluripotency when ectopically expressed in human fibroblasts in combination with NANOG, OCT4, and SOX2. Mammalian LIN28 functions in part by regulating processing of the let-7 microRNA through a GGAG binding site in the pre-let-7's distal loop region. However, many human and animal let-7 precursors lack the GGAG binding motif. In order to dissect the molecular mechanisms underlying its biological functions in a living animal, we identified a map of LIN-28 interactions with the transcriptome by in vivo HITS-CLIP in Caenorhabditis elegans. LIN-28 binds a large pool of messenger RNAs, and a substantial fraction of the bona fide LIN-28 targets are involved in aspects of animal development. Furthermore, our data show that LIN-28 regulates the expression of the let-7 microRNA by binding its primary transcript in a previously unknown region, revealing a novel regulatory mechanism.

The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42-37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer.