Jayanth Kumar Palanichamy

miRNA dysregulation in cancer: towards a mechanistic understanding

It is now well known that gene expression is intricately regulated inside each cell especially in mammals. There are multiple layers of gene regulation active inside a cell at a given point of time. Gene expression is regulated post-transcriptionally by microRNAs and other factors. Mechanistically, microRNAs are known to bind to the 3’ UTR of mRNAs and cause repression of gene expression and the number of known microRNAs continues to increase every day. Dysregulated microRNA signatures in different types of cancer are being uncovered consistently implying their importance in cellular homeostasis. However when studied in isolation in mouse models, clear-cut cellular and molecular mechanisms have been described only for a select few microRNAs. What is the reason behind this discrepancy? Are microRNAs small players in gene regulation helping only to fine tune gene expression? Or are their roles tissue and cell type-specific with single-cell level effects on mRNA expression and microRNA threshold levels? Or does it all come down to the technical limitations of high-throughput techniques, resulting in false positive results? In this review, we will assess the challenges facing the field and potential avenues for resolving the cellular and molecular mechanisms of these small but important regulators of gene expression.

Silencing of Integrated Human Papillomavirus-16 Oncogenes by Small Interfering RNA–Mediated Heterochromatization

Double-stranded RNAs or small interfering RNAs (siRNA) targeting the promoters of genes are known to cause gene knockdown by a process known as transcriptional gene silencing (TGS). We screened multiple siRNAs homologous to one of the NF-1 binding sites in the human papillomavirus-16 (HPV-16) enhancer and identified one siRNA which causes specific TGS of the HPV-16 oncogenes E6 and E7 when transfected into two HPV-16–positive cell lines siHa and CaSki. This phenomenon was specific to the HPV-16 enhancer with no effect on the HPV-18 enhancer. TGS was associated with heterochromatization of the targeted region of the enhancer but no DNA methylation was noted during the time period studied. The choice of target in the enhancer was important as siRNAs differing by one or two bases showed no suppression of downstream gene expression. A low copy number enhancer-associated transcript was detected in the cell lines studied and its level decreased significantly after treatment with the siRNA that caused TGS. This supports the RNA:RNA model described previously for TGS. This siRNA which causes simultaneous silencing of E6 as well as E7 oncogenes by an epigenetic mechanism might be useful as a therapeutic modality for HPV-16–positive cervical and other epithelial cancers. Mol Cancer Ther; 9(7); 2114–22. ©2010 AACR.

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia

BackgroundLong non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia.ResultsCASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter.ConclusionsOur findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

CpG Hypermethylation of the C-myc Promoter by dsRNA Results in Growth Suppression

Deregulation of the c-myc proto-oncogene plays an important role in carcinogenesis. It is, therefore, commonly found to be overexpressed in various types of tumors. Downregulation of c-myc expression assumes great importance in tumor therapy because of its ability to promote and maintain cancer stem cells. Apart from post-transcriptional gene silencing (PTGS), siRNAs have also been shown to cause transcriptional gene silencing (TGS) through epigenetic modifications of a gene locus. This approach can potentially be used to silence genes for longer periods and at a much lesser dosage than PTGS. In this study, we have examined the effect of transfection of a novel siRNA directed against a CpG island encompassing the CT-I(2) region in the P2 promoter of c-myc in U87MG and other cell lines. Transient transfection with this siRNA resulted in c-myc promoter CpG hypermethylation and decreased expression of c-myc (both mRNA and protein) and its downstream targets. A decrease was also observed in the expression of some stemness markers (oct-4 and nanog). Stable transfection also confirmed the promoter CpG hypermethylation and reduced c-myc expression along with reduced cell proliferation and an increase in apoptosis and senescence. A significant decrease in c-myc levels was also observed in three other cancer cell lines after transient transfection under similar conditions. Thus this novel siRNA has the capability of becoming an effective therapeutic tool in malignancies with overexpression of c-myc and may be of particular use in the eradication of recalcitrant cancer stem cells.

HIF-2α mediates a marked increase in migration and stemness characteristics in a subset of glioma cells under hypoxia by activating an Oct-4/Sox-2-Mena (INV) axis

Hypoxia is a salient feature of most solid tumors and plays a central role in tumor progression owing to its multiple contributions to therapeutic resistance, metastasis, angiogenesis and stemness properties. Reports exist in literature about hypoxia increasing stemness characteristics and invasiveness potential of malignant cells. In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies. We have demonstrated that U87MG and A172 glioma cells inherently have a subset of cells with high migratory potential due to migration-inducing Mena transcripts. These cells also have elevated stemness markers (Sox-2 and Oct-4). There was a significant increase of number in this subset of migratory cells on exposure to hypoxia with corresponding elevation (over 1000 fold) in migration-inducing Mena transcripts. We were able to demonstrate that a HIF-2α-Sox-2/Oct-4-Mena (INV) axis that is strongly activated in hypoxia and markedly increases the migratory potential of the cells. Such cells also formed tumor spheres with greater efficiency. We have correlated our in-vitro results with human glioblastoma samples and found that hypoxia, invasiveness and stemness markers correlated well in native tumor samples. This study identifies a novel signaling mechanism mediated by HIF-2α in regulating invasiveness and stemness characteristics, suggesting that under hypoxic conditions, some tumor cells acquire more migratory potential by increased Pan Mena and Mena INV expression as a consequence of this HIF-2α mediated increase in Oct-4 and Sox-2. These properties would help the cells to form a new nidus after local invasion or metastasis.

Survivin siRNA increases sensitivity of primary cultures of ovarian cancer cells to paclitaxel

PurposeThis aim of this study was to use ovarian cancer cells shed in ascitic fluid to establish primary cultures and subsequently use it to detect drug resistance to paclitaxel. Survivin siRNA was used to down regulate survivin expression and effect on paclitaxel resistance was also evaluated.MethodologyAscitic fluid along with corresponding primary tumor tissue was collected from twenty untreated epithelial ovarian cancer patients. Ten primary cultures were established from ascites obtained from untreated ovarian cancer patients in MCDB 105 and M199 medium (ratio 1:1). Knockdown of survivin was done using siRNA and sensitivity to paclitaxel was evaluated by MTT assay.ResultsGrape-like clusters of ovarian cancer cells present in ascites attached and gave a characteristic cobble stone appearance. Treatment with survivin siRNA resulted in a fivefold decrease in survivin expression in primary cultures. Survivin siRNA treatment significantly increased the sensitivity of the primary ovarian cancer cell cultures to paclitaxel.ConclusionAscitic cancer cells reflect the molecular profile of tumor and can be used to diagnose resistance to chemotherapy. This study also establishes that high survivin expression is also responsible for resistance to paclitaxel.

BALR-6 regulates cell growth and cell survival in B-lymphoblastic leukemia.

BackgroundA new class of non-coding RNAs, known as long non-coding RNAs (lncRNAs), has been recently described. These lncRNAs are implicated to play pivotal roles in various molecular processes, including development and oncogenesis. Gene expression profiling of human B-ALL samples showed differential lncRNA expression in samples with particular cytogenetic abnormalities. One of the most promising lncRNAs identified, designated B-ALL associated long RNA-6 (BALR-6), had the highest expression in patient samples carrying the MLL rearrangement, and is the focus of this study.ResultsHere, we performed a series of experiments to define the function of BALR-6, including several novel splice forms that we identified. Functionally, siRNA-mediated knockdown of BALR-6 in human B-ALL cell lines caused reduced cell proliferation and increased cell death. Conversely, overexpression of BALR-6 isoforms in both human and mouse cell lines caused increased proliferation and decreased apoptosis. Overexpression of BALR-6 in murine bone marrow transplantation experiments caused a significant increase in early hematopoietic progenitor populations, suggesting that its dysregulation may cause developmental changes. Notably, the knockdown of BALR-6 resulted in global dysregulation of gene expression. The gene set was enriched for leukemia-associated genes, as well as for the transcriptome regulated by Specificity Protein 1 (SP1). We confirmed changes in the expression of SP1, as well as its known interactor and downstream target CREB1. Luciferase reporter assays demonstrated an enhancement of SP1-mediated transcription in the presence of BALR-6. These data provide a putative mechanism for regulation by BALR-6 in B-ALL.ConclusionsOur findings support a role for the novel lncRNA BALR-6 in promoting cell survival in B-ALL. Furthermore, this lncRNA influences gene expression in B-ALL in a manner consistent with a function in transcriptional regulation. Specifically, our findings suggest that BALR-6 expression regulates the transcriptome downstream of SP1, and that this may underlie the function of BALR-6 in B-ALL.

Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region.

Background Recently various studies have demonstrated the role of promoter associated non-coding RNAs (pRNA) in dsRNA induced transcriptional gene silencing and activation. However the exact mechanistic details of these processes with respect to the orientation of pRNAs are poorly defined. Methodology/Principal Findings We have identified novel sense and antisense long control region (LCR) associated RNAs (pRNAs) in HPV18 positive cervical cancer cell lines HeLa, C-4 I and C-4 II. Using dsRNAs against these pRNAs, we were able to achieve upregulation or downregulation of the sense and antisense pRNAs and the downstream E6 and E7 oncogenes. We present evidence that knockdown of the sense pRNA is associated with reduction in E6 and E7 oncogenes and an upregulation of antisense pRNA. Conversely upregulation of sense pRNA is accompanied by an induction of the oncogenes and a concomitant reduction in antisense pRNA. Moreover, the exact role of sense and antisense pRNAs in dsRNA mediated gene modulation was confirmed by their selective degradation using antisense phosphorothioate oligodeoxynucleotides (ODN). Degradation of sense pRNA with antisense ODN led to loss of dsRNA induced silencing and activation, suggesting that dsRNA mediated gene modulation requires sense pRNA. Both processes were accompanied with congruent changes in the methylation pattern of activating and repressive histones. Conclusion/Significance Thus this data identifies and demonstrates the role of previously unknown important regulatory transcripts in HPV18 gene expression which can prove valuable targets in cervical cancer therapeutics. This mode of gene regulation by bidirectional transcription could be operational in other promoters as well and serve as a mechanism of regulating gene expression.

Transcriptional modulation of HIV-1C LTR promoter

Background All current anti-HIV1 therapies target the viral proteins or RNA; however targeting HIV1 at the transcriptional level of the integrated provirus has been less explored. In India, AIDS is commonly caused by HIV-1C compared to HIV-1B in developed countries. HIV1-5’LTR acts as a promoter and shows sequence variation among different clades. Transcriptional gene silencing (TGS) is a method wherein dsRNA targeting the promoter/ enhancer of a gene are used to down regulate its expression.