LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans.

Colorectal cancer (CRC) remains a major contributor to cancer-related mortality. LIN28A and LIN28B are highly related RNA-binding protein paralogs that regulate biogenesis of let-7 microRNAs and influence development, metabolism, tissue regeneration, and oncogenesis. Here we demonstrate that overexpression of either LIN28 paralog cooperates with the Wnt pathway to promote invasive intestinal adenocarcinoma in murine models. When LIN28 alone is induced genetically, half of the resulting tumors harbor Ctnnb1 (ß-catenin) mutation. When overexpressed in Apc(Min/+) mice, LIN28 accelerates tumor formation and enhances proliferation and invasiveness. In conditional genetic models, enforced expression of a LIN28-resistant form of the let-7 microRNA reduces LIN28-induced tumor burden, while silencing of LIN28 expression reduces tumor volume and increases tumor differentiation, indicating that LIN28 contributes to tumor maintenance. We detected aberrant expression of LIN28A and/or LIN28B in 38% of a large series of human CRC samples (n = 595), where LIN28 expression levels were associated with invasive tumor growth. Our late-stage CRC murine models and analysis of primary human tumors demonstrate prominent roles for both LIN28 paralogs in promoting CRC growth and progression and implicate the LIN28/let-7 pathway as a therapeutic target.

Retinoic acid signalling regulates the development of tonotopically patterned hair cells in the chicken cochlea.

Precise frequency discrimination is a hallmark of auditory function in birds and mammals and is required for distinguishing similar sounding words, like 'bat,' 'cat' and 'hat.' In the cochlea, tuning and spectral separation result from longitudinal differences in basilar membrane stiffness and numerous individual gradations in sensory hair cell phenotypes, but it is unknown what patterns the phenotypes. Here we used RNA-seq to compare transcriptomes from proximal, middle and distal regions of the embryonic chicken cochlea, and found opposing longitudinal gradients of expression for retinoic acid (RA)-synthesizing and degrading enzymes. In vitro experiments showed that RA is necessary and sufficient to induce the development of distal-like hair cell phenotypes and promotes expression of the actin-crosslinking proteins, Espin and Fscn2. These and other findings highlight a role for RA signalling in patterning the development of a longitudinal gradient of frequency-tuned hair cell phenotypes in the cochlea.

The transcriptome of utricle hair cell regeneration in the avian inner ear.

Sensory hair cell loss is the major cause of hearing and balance disorders. Mammals are incapable of sustained hair cell regeneration, but lower vertebrates can regenerate these mechano-electrical transducers. We present the first comprehensive transcriptome (by mRNA-Seq) of hair cell regeneration in the chick utricle. We provide pathway and pattern annotations and correlate these with the phenotypic events that occur during regeneration. These patterns are surprisingly synchronous and highly punctuated. We show how these patterns are a new resource for identifying components of the hair cell transcriptome and identify 494 new putative hair-cell-specific genes and validate three of these (of three tested) by immunohistochemical staining. We describe many surprising new components and dynamic expression patterns, particularly within NOTCH signaling. For example, we show that HES7 is specifically expressed during utricle hair cell regeneration and closely parallels the expression of HES5. Likewise, the expression of ATOH1 is closely correlated with HEYL and the HLH inhibitory transcription factors ID1, ID2, and ID4. We investigate the correlation between fibroblast growth factor signaling and supporting cell proliferation and show that FGF20 inhibits supporting cell proliferation. We also present an analysis of 212 differentially expressed transcription factor genes in the regenerative time course that fall into nine distinct gene expression patterns, many of which correlate with phenotypic events during regeneration and represent attractive candidates for future analysis and manipulation of the regenerative program in sensory epithelia and other vertebrate neuroepithelia.

Combined deep microRNA and mRNA sequencing identifies protective transcriptomal signature of enhanced PI3Kα signaling in cardiac hypertrophy.

The perturbation of myocardial transcriptome homeostasis is the hallmark of pathological hypertrophy, underlying the maladaptive myocardial remodeling secondary to pathological stresses. Classic and novel therapeutics that provide beneficial effects against pathological remodeling likely impact myocardial transcriptome architecture, including miRNA and mRNA expression profiles. Microarray and PCR-based technologies, although employed extensively, cannot provide adequate sequence coverage or quantitative accuracy to test this hypothesis directly. The goal of this study was to develop and exploit next-generation sequencing approaches for comprehensive and quantitative analyses of myocardial miRNAs and mRNAs to test the hypothesis that augmented phosphoinositide-3-kinase-p110α (PI3Kα) signaling in the setting of pathological hypertrophy provides beneficial effects through remodeling of the myocardial transcriptome signature. In these studies, a molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kα signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored. These analyses identified multiple miRNAs and mRNAs that were abnormally expressed in pathological hypertrophy and partially or completely normalized with increased PI3Kα signaling. Additionally, several novel miRNAs potentially linked to remodeling in cardiac hypertrophy were identified. Additional experiments revealed that increased PI3Kα signaling reduces cardiac fibrosis in pathological hypertrophy through modulating TGF-ß signaling and miR-21 expression. In conclusion, using the approach of combined miRNA and mRNA sequencing, we identify the protective transcriptome signature of enhanced PI3Kα signaling in the context of pathological hypertrophy, and demonstrate the regulation of TGF-ß/miR-21 by which enhanced PI3Kα signaling protects against cardiac fibrosis.

A cross-species analysis of microRNAs in the developing avian face.

Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27(KIP1) is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development.

An RNA interference-based screen of transcription factor genes identifies pathways necessary for sensory regeneration in the avian inner ear.

Sensory hair cells of the inner ear are the mechanoelectric transducers of sound and head motion. In mammals, damage to sensory hair cells leads to hearing or balance deficits. Nonmammalian vertebrates such as birds can regenerate hair cells after injury. In a previous study, we characterized transcription factor gene expression during chicken hair cell regeneration. In those studies, a laser microbeam or ototoxic antibiotics were used to damage the sensory epithelia (SE). The current study focused on 27 genes that were upregulated in regenerating SEs compared to untreated SEs in the previous study. Those genes were knocked down by siRNA to determine their requirement for supporting cell proliferation and to measure resulting changes in the larger network of gene expression. We identified 11 genes necessary for proliferation and also identified novel interactive relationships between many of them. Defined components of the WNT, PAX, and AP1 pathways were shown to be required for supporting cell proliferation. These pathways intersect on WNT4, which is also necessary for proliferation. Among the required genes, the CCAAT enhancer binding protein, CEBPG, acts downstream of Jun Kinase and JUND in the AP1 pathway. The WNT coreceptor LRP5 acts downstream of CEBPG, as does the transcription factor BTAF1. Both of these genes are also necessary for supporting cell proliferation. This is the first large-scale screen of its type and suggests an important intersection between the AP1 pathway, the PAX pathway, and WNT signaling in the regulation of supporting cell proliferation during inner ear hair cell regeneration.

Complex mutation patterns of epidermal growth factor receptor gene associated with variable responses to gefitinib treatment in patients with non-small cell lung cancer.

Mutational analysis was performed in the kinase domain (exons 18-21) of the EGFR gene on tumor tissues of 65 non-small cell lung cancer (NSCLC) patients who had received gefitinib monotherapy. The association between EGFR gene mutation, gefitinib treatment response, and the overall survival were evaluated. In total, EGFR mutations with complex patterns were identified in 32 tumors. The overall mutation rate was 49.2% (32/65). Twenty of the 32 patients were responders, 10 non-responders, and 2 not assessable. The most common mutation in non-responders was L858R. Gefitinib responsiveness was only significantly associated with EGFR mutation and adenocarcinoma. The median survival for responder (15.5 months) was much longer than non-responder (9.23 months), though the difference only had marginal significance (p=0.056). The difference of overall survival between patients with and without EGFR mutation was non-significant (p=0.7819), mainly due to the short survival of the non-responders with EGFR mutations (median survival=6.2 months). Our study revealed that the response to gefitinib treatment in NSCLC patients with EGFR mutations could be quite variable even for the same EGFR mutation type. An analysis of the various EGFR mutations and the response patterns was also performed and compared with recently published reports on EGFR mutation and gefitinib responsiveness.

High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan.

PURPOSE: Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non-small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non-small cell lung cancer. EXPERIMENTAL DESIGN: Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues. RESULTS: The 101 non-small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non-small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations. CONCLUSIONS: Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.