Emily J. Noonan

MicroRNA-373 induces expression of genes with complementary promoter sequences

Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.

Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function

Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid hormone receptor family and is very important for the development and progression of prostate cancer. The soy isoflavone genistein has been shown previously to down-regulate AR in androgen-dependent prostate cancer cell lines such as LNCaP. However, the mechanism(s) by which AR is down-regulated by genistein is still not known fully. We show a new mechanism by which genistein inhibits AR protein levels. We show that genistein-treated LNCaP cells exhibit increased ubiquitination of AR, suggesting that AR protein is down-regulated via a proteasome-mediated pathway. AR is normally stabilized by the chaperone activity of the heat shock protein Hsp90. The increased ubiquitination of AR after genistein treatment is attributed to decreased Hsp90 chaperone activity as assessed by its increased functionally inactive acetylated form. Consistent with this result, we find that HDAC6, which is a Hsp90 deacetylase, is inhibited by the antiestrogenic activity of genistein. Hence, in this study, we elucidate a novel mechanism of AR down-regulation by genistein through inhibition of HDAC6-Hsp90 cochaperone function required to stabilize AR protein. Our results suggest that genistein could be used as a potential chemopreventive agent for prostate cancers along with known inhibitors of HDAC6 and Hsp90. [Mol Cancer Ther 2008;7(10):3195–202]

Hsp70B′ regulation and function

Abstract Heat shock protein (Hsp) 70B′ is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B′ and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B′. Flow cytometry was used to assay hsp70B′ promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B′ and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B′ is a secondary responder. Interestingly ZnSO4 induces Hsp70B′ and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B′. Both Hsp70B′ and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B′ contributes to cell survival.

HDAC3 overexpression and colon cancer cell proliferation and differentiation.

An immunohistochemical analysis of human colorectal adenocarcinomas showed that cancer cells express widely varying levels of HDAC3. The SW480 colon cancer cell line was found to express high levels of HDAC3 compared to other colon cancer cell lines. p21 was poorly induced in SW480 cells relative to the lower HDAC3‐expressing HT‐29 cells. RNAi‐induced reduction of HDAC3 in SW480 cells increased their constitutive, butyrate‐, TSA‐, and TNF‐α‐induced expression of p21, but did not cause all the gene expression changes induced upon general histone deacetylase (HDAC) inhibition. SW480 cells with lower HDAC3 expression appeared to be poised for gene expression responses with increased histone H4‐K12 acetylation, but not K5, K8, or K16 acetylation. Even though p21 was readily activated in HT29 cells, HDAC3 siRNA nonetheless stimulated p21 expression in these cells to a greater degree than HDAC1 and HDAC2 siRNA. SW480 cells with lower HDAC3 levels displayed an enhanced cell cycle arrest and growth inhibition by butyrate, but without changes in apoptosis or sensitivity to chemotherapeutic agents. As reported for other colon cancer cell lines, butyrate induced the rapid downregulation of the secretory cell differentiation markers mucin 2 and intestinal trefoil factor in SW480 cells. Interestingly, selective HDAC3 inhibition was sufficient to downregulate these genes. Our data support a central role for HDAC3 in regulating the cell proliferation and differentiation of colon cancer cells and suggest a potential mechanism by which colon cancers may become resistant to luminal butyrate.

Defining features and exploring chemical modifications to manipulate RNAa activity.

RNA interference (RNAi) is an evolutionary conserved mechanism by which small double-stranded RNA (dsRNA)--termed small interfering RNA (siRNA)--inhibit translation or degrade complementary mRNA sequences. Identifying features and enzymatic components of the RNAi pathway have led to the design of highly-effective siRNA molecules for laboratory and therapeutic application. RNA activation (RNAa) is a newly discovered mechanism of gene induction also triggered by dsRNAs termed small activating RNA (saRNA). It offers similar benefits as RNA interference (RNAi), while representing a new method of gene overexpression. In the present study, we identify features of RNAa and explore chemical modifications to saRNAs that improve the applicability of RNAa. We evaluate the rate of RNAa activity in order to define an optimal window of gene induction, while comparing the kinetic differences between RNAa and RNAi. We identify Ago2 as a conserved enzymatic component of both RNAa and RNAi implicating that saRNA may tolerate modification based on Ago2 function. As such, we define chemical modifications to saRNAs that manipulate RNAa activity, as well as exploit their effects to design saRNAs with enhanced medicinal properties. These findings reveal functional features of RNAa that may be utilized to augment saRNA function for mechanistic studies or the development of RNAa-based drugs.

HDACs and the senescent phenotype of WI-38 cells

BackgroundNormal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.ResultsPre- and post-senescent WI-38 cells were treated with the HDAC inhibitors butyrate or trichostatin A (TSA). Following HDAC inhibitor treatment, pre-senescent cells increased p21WAF1 and β-galactosidase expression, assumed a flattened senescence-associated morphology, and maintained a lower level of proteasome activity. These alterations also occurred during normal replicative senescence of WI-38 cells, but were not accentuated further by HDAC inhibitors. We also found that HDAC1 levels decline during normal replicative senescence.ConclusionOur findings indicate that HDACs impact numerous phenotypic changes associated with cellular senescence. Reduced HDAC1 expression levels in senescent cells may be an important event in mediating the transition to a senescent phenotype.

Non-coding RNAs turn up the heat: An emerging layer of novel regulators in the mammalian heat shock response

The field of non-coding RNA (ncRNA) has expanded over the last decade following the discoveries of several new classes of regulatory ncRNA. A growing amount of evidence now indicates that ncRNAs are involved even in the most fundamental of cellular processes. The heat shock response is no exception as ncRNAs are being identified as integral components of this process. Although this area of research is only in its infancy, this article focuses on several classes of regulatory ncRNA (i.e., miRNA, lncRNA, and circRNA), while summarizing their activities in mammalian heat shock. We also present an updated model integrating the traditional heat shock response with the activities of regulatory ncRNA. Our model expands on the mechanisms for efficient execution of the stress response, while offering a more comprehensive summary of the major regulators and responders in heat shock signaling. It is our hope that much of what is discussed herein may help researchers in integrating the fields of heat shock and ncRNA in mammals.

Cell number-dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

Hsp70B′ is a unique member of the human Hsp70 family of chaperones about which information is scarce. Unlike the major inducible Hsp72 protein, Hsp70B′ is strictly inducible having little or no basal expression levels in most cells. We observed that Hsp70B′ appears transiently in response to heat stress whereas Hsp72 levels persist for many days. Also, Hsp70B′ is optimally induced when cell numbers are low, whereas Hsp72 levels are greatest at higher cell number. Hsp70B′ promoter activation was measured by flow cytometry using an Hsp70B′ promoter‐driven GFP construct. In heat stressed cells, promoter activation is cell number independent over a broad range. However, when cell number increases beyond a certain population size, cells are less stress inducible for Hsp70B′ and induction becomes highly cell number‐dependent. Cell number differences in Hsp70 activation cannot be explained by changes in Hsf‐1 DNA‐binding activity or hyperphosphorylation. Cells with few or no cell matrix attachments (laminin‐coated and low attachment plates, respectively) appear to be more sensitive to cell number‐dependent inhibition. Medium conditioned by the low cell number (LCN) populations supports increased Hsp70B′ promoter activation in high cell number (HCN) cultures. Likewise, medium conditioned in HCN culture conditions causes decreased activation of Hsp70B′ promoter in LCN cultures. As HCN‐conditioned medium has all the components necessary for cell growth, two possibilities for the activation of Hsp70B′ gene expression exist: an inhibitory component that accumulates in culture medium at HCN, or an activator that accumulates at LCN. J. Cell. Physiol. 210: 201–211, 2007.

Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma

Small double stranded RNAs (dsRNA) are a new class of molecules which regulate gene expression. Accumulating data suggest that some dsRNA can function as tumor suppressors. Here, we report further evidence on the potential of dsRNA mediated p21 induction. Using the human renal cell carcinoma cell line A498, we found that dsRNA targeting the p21 promoter significantly induced the expression of p21 mRNA and protein levels. As a result, dsP21 transfected cells had a significant decrease in cell viability with a concomitant G1 arrest. We also observed a significant increase in apoptosis. These findings were associated with a significant decrease in survivin mRNA and protein levels. This is the first report that demonstrates dsRNA mediated gene activation in renal cell carcinoma and suggests that forced over‐expression of p21 may lead to an increase in apoptosis through a survivin dependent mechanism.

Surface expression of Hsp70B' in response to proteasome inhibition in human colon cells.

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.