Hit Validation

RNAx employ RNAi screening of drug sensitising genes in skin cancer research.

Here is how we can help you to get results.

RNA interference has become a widely used research tool. Increasingly, clinicians feel the need to use this method, but often lack the expertise, equipment and personnel to run RNAi screens. RNAx introduces to you a short synopsis of how we could help our colleagues at the Charité to conduct a RNA interference screen for essential and drug sensitizing genes in skin cancer cells.

RNAi Interference and Skin Cancer

We show that RNA interference can be used in melanoma cell lines to specifically inhibit gene expression very efficiently. By screening an apoptosis related library, factors with significant impact on cell viability could be identified, proving the reliability of such an approach. The combination of RNAi with chemotherapeutic drug treatment helped to discover drug sensitizing genes. By using four independent siRNAs per primary hits, phenotypes for 18 genes could be positively validated. Due to the promising results, the study will be expanded to genome wide scale.

Skin cancers (melanoma, epithelial skin tumours and cutaneous lymphomas) are among the major cancers affecting large parts of the populations with some of the highest increases in incidence rates. Most forms of epithelial skin tumours are not immediately life-threatening. However, they have the highest incidence rates of all human cancers and about 100 per 100,000 individuals are diagnosed each year for malignant forms of these skin cancers. Along with the aging of the European population, this rate is steadily, for some entities dramatically, rising.

Like melanoma, non-melanoma skin cancers, to an ever greater extend, also affect the younger populations, probably due to environmental changes. The therapeutic options for skin cancers are limited and palliative in most cases. In fact, whereas very early stages of melanoma and BCC can be surgically eliminated and some precancerous in situ (field) lesions of epithelial skin cancers cured by immune-modulating treatment, there is no curative therapy for manifest skin cancers of any of the three classes.

New concepts such as antibody-based therapies for cutaneous lymphomas, cytokines for lymphomas and melanoma, immunomodulating therapies for epithelial skin cancers and small molecular inhibitors of various enzymes in melanoma and cutaneous lymphoma are in clinical trials. However, none of these new therapies were primarily developed for skin cancers but rather translated from other cancer into the skin cancer field. The efficacy of these new therapies so far appears limited although the new therapeutic principles of these therapies justify high expectations for future breakthroughs.

The discovery and use of RNA interference (RNAi) is a revolution in molecular and cellular biology that enables loss-of-function studies on genome scale. RNAi is a naturally occurring mechanism by which small interfering RNAs (siRNAs), double stranded RNAs of 21-23 base pairs, when incorporated into RNA-induced silencing complex (RISC), guide degradation of RNAs with sequences identical or almost identical to the siRNA. While cells use RNA silencing for protection against virus infections, to keep transposons in check, and, in case of the microRNAs (miRNAs), to control development, experimental RNAi mainly aims to knock down specific proteins expression for functional studies.

RNAx, a spin-off from the Max-Planck-Institute for infection biology, has set up an automated RNA interference platform for si/shRNA validation and functional genome analysis.

RNAi Screening

In order to gain a deeper understanding of skin cancers and to identify new therapeutic targets, for the group of Professor Peter Walden in the department of Dermatology, Venerology and Allergy at the Charité - University Medicine in Berlin, the platform has been used to perform RNAi screening to identify essential as well as drug sensitizing genes in melanoma cells.

To establish appropriate cellular models for the identification of new drug targets, three melanoma cell lines were analysed for their susceptibility to siRNA transfection. As shown in Figure 1, in all three cell lines the expression levels of the target mRNA could be suppressed by more than 97%, ensuring that RNAi based loss-of-function approaches are feasible in melanoma cells.

RNA Interference
Figure 1: RNA interference in melanoma cells.
The indicated melanoma cell lines have been transfected either with a non-silencing siRNA or a NKEFA directed siRNA. Three days post transfection, RNA has been purified from the cells and subjected to real time RT-PCR measurement of the target gene and an internal standard gene (GAPDH). Target gene expression has been normalized against the reference transfected cells and the expression level of the internal standard gene. Error bars indicate standard deviations.

Next, the effect of knockdown of PLK1 and SKIP, which was shown to be toxic in various other cell models was tested for the three model systems. For this we made use of a microscopic readout, where we counted fluorescently labelled nuclei. As shown in figure 2 for ChaMel84 cells, knockdown of both model genes lead to either almost complete loss of cells or to 60% reduction of cell number.

Gene Knockdown Effects
Figure 2: Cytotoxic effect of gene knockdown.
ChaMel84 cells have been transfected with siRNAs against PLK1 and SKIP. Allstars (Qiagen) siRNA was used as a transfection control formerly shown to have no effect on ChaMel84 cell viability. 7 days after transfection, cells have been fixed. Nuclei were stained with Hoechst dye 33258 and counted by an automated Scan^R microscope system from Olympus.

A promising approach to treat cancer is to combine several drugs exerting synergistic effects. For this purpose, cells have been treated with the chemotherapeutic drug Etoposide, leading to a nearly 50% reduction of cells in allstars, non-silencing control samples. In contrast to this, reduction of cell number was less prominent in samples where gene knockdown itself was already toxic.
In a pilot study to prepare for a genome wide screen, a library targeting 418 apoptosis related genes (Qiagen) was screened. For this, two siRNAs targeting each gene where pooled into one well.

The screening experiments were repeated three times independently. Cell numbers of non-treated and treated samples of each plate were normalised against the mean values of four allstars-treated samples. We could identify 40 genes reducing cell number by more than 65% and 9 genes, whose knockdown increases cell number by more than 15%. The statistical significance of the phenotypes were proven by student's T-test. In addition, knockdown of 15 genes exert significant reductions of cell numbers only after Etoposide induction, providing first hints for synergistic effects of combined drug treatments. In cases where Etoposide treatment had no effect on cell numbers at all, important factors for Etoposide-induced cell death might have been hit.

Gene Screening
Figure 3: Screen for genes essential for ChaMel84 cell survival.
ChaMel84 cells have been transfected with siRNAs against 418 apoptosis related genes. Four days after transfection, cells have either been treated with 20 µM Etoposide (Sigma) or not. Three days post induction, cells have been fixed. Nuclei were stained with Hoechst dye 33258 and counted by an automated Scan^R microscope system from Olympus. Cell numbers have been normalized to untreated Allstars transfected samples for each plate separately. Untreated samples are shown In blue, Etoposide treated samples in red.

Hit Validation

It is well known that siRNAs can exert off-target effects. These are mainly due to three major reasons:

1) Activation of interferon responses
2) Knockdown of mRNAs with sequence homologies to the intended target
3) Displacement of endogenous miRNAs from RISC by the transfected siRNA leading to the abolishment of miRNA functions.

In order to sort out primary hits related to off-target effects, we repeated the experiments with four additional siRNAs per putative target. As expected for a large number of the hits, primary phenotypes could not be confirmed.

Hit Validation
Figure 4: Hit validation
ChaMel84 cells have been transfected with four independent siRNAs against each primary hit. Four days after transfection, cells have either been treated with 20 µM Etoposide (Sigma) or not. Three days post induction, cells have been fixed. Nuclei were stained with Hoechst dye 33258 and counted by an automated Scan^R microscope system from Olympus. Cell numbers have been normalized to untreated Allstars transfected samples for each plate separately. As controls siRNAs against PLK1 and SNW have been used.

However for nine genes whose knockdown led to reduced cell numbers in the primary screen, with two or more out of four siRNAs, the phenotypes could be reproduced. Furthermore, for three genes, increased cell numbers after knockdown could be seen with at least two out of four siRNAs. Additionally, knockdown of six genes sensitized cells for Etoposide underscoring the reliability of the screening approach to identify targets for combinatorial cancer therapies.

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