Olivier Loudig

Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483-1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10(-8) M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARgamma (RA receptor gamma)/RXRalpha (retinoid X receptor alpha) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumors invasive properties.

Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155*.

Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNβ production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus‐mediated IRF3 gene transfer (Ad‐IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL‐1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti‐inflammatory one, akin to an M1–M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine‐induced death in glial‐neuronal co‐cultures. Furthermore, Ad‐IRF3 suppressed the expression of microRNA‐155 and its star‐form partner miR‐155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR‐155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR‐155/miR‐155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression.

Hsa‐miR‐375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity

Invasive lobular carcinoma (ILC) of the breast, characterized by loss of E‐cadherin expression, accounts for 5–15% of invasive breast cancers and it is believed to arise via a linear histological progression. Genomic studies have identified a clonal relationship between ILC and concurrent lobular carcinoma in situ (LCIS) lesions, suggesting that LCIS may be a precursor lesion. It has been shown that an LCIS diagnosis confers a 15–20% risk of progression to ILC over a lifetime. Currently no molecular test or markers can identify LCIS lesions likely to progress to ILC. Since microRNA (miRNA) expression changes have been detected in a number of other cancer types, we explored whether their dysregulation might be detected during progression from LCIS to ILC. Using the Illumina miRNA profiling platform, designed for simultaneous analysis of 470 mature miRNAs, we analysed the profiles of archived normal breast epithelium, LCIS lesions found alone, LCIS lesions concurrent with ILC, and the concurrent ILCs as a model of linear histological progression towards ILC. We identified two sets of differentially expressed miRNAs, the first set highly expressed in normal epithelium, including hsa‐miR‐224, ‐139, ‐10b, ‐450, 140, and ‐365, and the second set up‐regulated during lobular neoplasia progression, including hsa‐miR‐375, ‐203, ‐425‐5p, ‐183, ‐565, and ‐182. Using quantitative RT‐PCR, we validated a trend of increasing expression for hsa‐miR‐375, hsa‐miR‐182, and hsa‐miR‐183 correlating with ILC progression. As we detected increased expression of hsa‐miR‐375 in LCIS lesions synchronous with ILC, we sought to determine whether hsa‐miR‐375 might induce phenotypes reminiscent of lobular neoplasia by expressing it in the MCF‐10A 3D culture model of mammary acinar morphogenesis. Increased expression of hsa‐miR‐375 resulted in loss of cellular organization and acquisition of a hyperplastic phenotype. These data suggest that dysregulated miRNA expression contributes to lobular neoplastic progression. Copyright

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

BackgroundAxl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.MethodsWT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freunds adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays.ResultsAxl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals.ConclusionsThese data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.

Background Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses. Significance We determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Loss of retinal cadherin facilitates mammary tumor progression and metastasis

The mammary epithelium is thought to be stabilized by cell-cell adhesion mediated mainly by E-cadherin (E-cad). Here, we show that another cadherin, retinal cadherin (R-cad), is critical for maintenance of the epithelial phenotype. R-cad is expressed in nontransformed mammary epithelium but absent from tumorigenic cell lines. In vivo, R-cad was prominently expressed in the epithelium of both ducts and lobules. In human breast cancer, R-cad was down-regulated with tumor progression, with high expression in ductal carcinoma in situ and reduced expression in invasive duct carcinomas. By comparison, E-cad expression persisted in invasive breast tumors and cell lines where R-cad was lost. Consistent with these findings, R-cad knockdown in normal mammary epithelium stimulated invasiveness and disrupted formation of acini despite continued E-cad expression. Conversely, R-cad overexpression in aggressive cell lines induced glandular morphogenesis and inhibited invasiveness, tumor formation, and lung colonization. R-cad also suppressed the matrix metalloproteinase 1 (MMP1), MMP2, and cyclooxygenase 2 gene expression associated with pulmonary metastasis. The data suggest that R-cad is an adhesion molecule of the mammary epithelium, which acts as a critical regulator of the normal phenotype. As a result, R-cad loss contributes to epithelial suppression and metastatic progression.

Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

Gene expression profiling of formalin-fixed and paraffin-embedded (FFPE) specimens, banked from completed clinical trials and routine clinical care, has the potential to yield valuable information implicating and linking genes with clinical parameters. In order to prepare high-quality cDNA from highly fragmented FFPE-RNA, previously precluded from high-throughput analyses, we have designed a novel strategy based on the nucleic acid restoration of incomplete cDNA sequences prior to T7 in vitro transcription (IVT) amplification. We describe this strategy as complementary-template reverse-transcription (CT-RT) because short single-stranded T7-oligo-dT24-VN-DNA sequences, obtained from FFPE-RNA, are used as primers for the RT of complementary RNA templates contained in a sense-RNA library. We validated our assay by determining the correlation between expression profiles of a matched 10-year-old frozen and FFPE breast cancer sample. We show that T7 IVT-amplification of cDNA transcripts restored by CT-RT is a specific and reliable process that allows recovery of transcriptional features undetectable by direct T7 IVT-amplification of FFPE-RNA. Furthermore, CT-RT restored 35–41% of the transcripts from archived breast and cervical specimens when compared to matched frozen tissue; and profiles included tissue-specific transcripts. Our results indicate that CT-RT allows microarray profiling of severely degraded RNA that could not be analyzed by previous methods.

N-cadherin regulates mammary tumor cell migration through Akt3 suppression.

N-cadherin is a cell–cell adhesion molecule that plays a role in breast cancer metastasis. Here, we show that in vivo expression of N-cadherin in the PyMT mouse model, which enhances mammary tumor metastasis, results in selective inhibition of Akt3 expression and phosphorylation. Similarly, exogenous expression of N-cadherin in PyMT or MCF-7 mammary tumor cells enhanced cell motility and caused a dramatic reduction in Akt3 expression and phosphorylation. Moreover, knockdown of Akt3 in PyMT tumor cells increased cell motility and disrupted mammary morphogenesis, but had no effect on cell proliferation. Conversely, overexpression of wild-type Akt3 in PyMT-N-cadherin cells inhibited cell motility promoted by N-cadherin. Taken altogether, these findings demonstrate that N-cadherin suppresses Akt3 to promote cell motility and highlight the intricate regulation of Akt isoforms by N-cadherin during metastasis.

MicroRNAs are expressed and processed by human primary macrophages.

Macrophages are crucial to host defense, functioning in innate and cell-mediated immunity. MicroRNAs (miRNAs) are small non-coding RNA molecules that repress transcription and protein production. Little is known about miRNA expression in primary human macrophages, or about how macrophage miRNAs contribute to both normal macrophage function and to the pathogenesis of disease in humans. Using Western blot analyses, we demonstrated the production of miRNA machinery proteins by human primary macrophages. Using two different miRNA array techniques, we identified 119 miRNAs expressed by human primary macrophages, including hsa-let-7a, miR-16, -23a, 30b, -103, -146a, -212, and -378 and validated them by quantitative RT-PCR. Our findings provide a knowledge base to which macrophage miRNA expression in organ-specific macrophages or disease processes may be compared in humans.