Functional Genomics and Marker Validation

Aims  and Vision

The aim of “Functional Genomics” in MOMA is a functional characterization of new cancer biomarkers and the investigation of their mode of action as well as their pathway involvement. This will result in a better understanding of the molecular mechanisms triggering the development and progression of cancer.

Our vision is the functional characterization of potential new diagnostic or prognostic biomarkers or potential therapy targets and the survey of their potential usability in clinics. Moreover, our vision is to map the complex intracellular network controlling cellular behaviour as e.g. invasive growth or formation of metastases.
All these issues shall address a more rational treatment for patients and will thus contribute to a tailor suited therapy for cancer patients in the future.

Current Research Activities 

On the one hand, signatures will be validated in large multicenter retrospective and prospective studies with the aim to use theses signatures for clinical application (see organ sites). On the other hand, functional analyses will be performed on selected molecules with clinical relevance. Those molecules comprise e.g. mRNA transcripts encoding proteins as well as microRNAs.

MOMA’s functional characterizations  

• single molecules from signatures predicting disease outcome, comprising molecules characterizing recurrence, progression or carcinoma in situ (CIS) in bladder cancer.
• single molecules from a signature predicting response to various forms of chemotherapy in bladder cancer.
• key microRNAs in bladder carcinomas with regard to progression, CIS (carcinoma in situ) and chemotherapy response.
• splice variants expressed in different bladder cancer stages or different colon cancer stages .
• molecules from signatures capable of predicting clinically relevant endpoints like recurrence of disease and response to colon cancer treatment.
• microRNAs with prognostic potential, especially in relation to the prediction of metastatic recurrence of coloncancer .

Research Strategy



Each project has its origin in the box headed “Biobank”, where samples are collected and nucleic acids are extracted, quality controlled and stored (for detailed information, see CRC or bladder cancer page).
According to the aim of the project, samples will be selected/collected based on statistical power calculations according to availability of material. Samples are profiled and data are subjected to bioinformatic analyses. Candidates are validated by RTPCR.

Expression of gene encoded proteins and their localization within the tumor cell is analyzed by immunohistochemistry using cryosections in Tissue Tek or FFPE tissue specimen.






 

 

For protein detection, antibody specificity is checked on Western Blots using cell lines overexpressing the molecule of interest.

(Birkenkamp-Demtröder et al, Mol Onc, 2007)




In situ hybridization (ISH) using LNA probes is used to localize microRNAs within the tumor cells.



	(pictures with permission of Zeiss/Brock&Michelsen, DK)
After optimization, Tissue microarrays (TMA’s) with cores from a large number of tissue samples with detailed 5 years clinical follow-up data are stained with the specific antibody to identify a clinical correlation with disease free survival.	TMA slides are scanned by a Mirax scanner or a Hamamatsu scanner and antibody staining is scored using 3D-Histech or Visiopharm imaging software.

 
To decide which clinical relevant molecule should be further analysed, intensive literature searches has to be done investigating matters like function, localization, pathway involvement, drugability or availability of animal models.

Selected candidate genes are cloned and overexpressed in mammalian cancer cell lines with known expression pattern or knocked down by Small interference RNA (siRNA) mediated silencing.

Cell Line authentication

We are working with several different cell lines from colon, bladder and prostate as well as human cells as HELA or HEK cell lines. Within regular intervals, cell lines are tested for mycoplasma and are re-authenticated via STR – short tandem repeat analysis  (Masters JR et al, Proc Natl Acad Sci U S A 2001;98:8012-7) using the Cell-ID-system G9500 from Promega. Products are analyzed on an Applied-Biosystems 3130 Genetic Analyzer.

In case of microRNAs cells are transfected with miR-precursors or Anti-miRs, miRNA Inhibitors targeting human miRNAs (Exiqon). Successful up- or downregulation is monitored by RTPCR (Taqman) and Western blotting for protein.





Cells with an overexpressed or silenced molecule of interest are continuously monitored by Real Time Cell Analysis RTCA (Roche X-celligence) over a period of up to 5 days to identify whether the molecule of interest impacts cellular processes, e.g. increases proliferation or causes cell death. Standard cellular assays monitoring proliferation, viability or cytotoxicity are performed.

 

 



 

 

Data analyses in MOMA are performed on a Dual Plate RTCA instrument, offering highest possible flexibility for several different approaches in parallel.







(with permission of Roche Diagnostics, DK)

 

 

Fluorescence microscopy visualizes the cellular localization of proteins within living or fixed cells from cell lines, from cryosections or from FFPE tissue samples.

Co-localization (yellow) of KRT23 protein (green) with Golgi Marker 58K (red) in the Golgi-apparatus as identified in KRT23 overexpressing cells as well as in a solid colon adenocarcinoma. (Mansilla et al, J.Mol med, 2008)	Co-localization of LPCAT1 protein (green) with mitochondrial marker Prohibitin (red) identified in LPCAT1 overexpressing cells. (Birkenkamp-Demtröder et al, Mol Onc, 2007)

 

	For 3D-microimaging analyses, we are using a ZEISS Axiovision 200M microscope with apotome.
(with permission from Zeiss/Brock&Michelsen, DK)	The application of an apotome results in an optical sectioning of the preparates, which is comparable to confocal lasermicroscopy.

In addition to the above mentioned analyses as e.g. RTCA, protein work and microscopy, a large number of our functional cellular analyses are performed in 24-96 well microtiterplates using a Biotek Synergy HT Microplatereader.
This instrument supports assays based on luminescence, fluorescence or absorbance. Our analyses comprise studies on proliferation, viability, invasion, migration, adhesion, cell death and characterization of apoptosis of manipulated versus mock treated cells.




For Pathway analyses, to gain insight into networks or to identify downstream target molecules, cell extracts from manipulated cells, overexpressing a molecule of interest or with a knock down, are subjected to Microarray expression profiling. Bioinformatic data processing is followed by application of Ingenuity Pathway analyses software to identify pathways or networks addressed in common for both, the tumor samples as well as the manipulated cell lines. (K. Demtröder, unpublished data)
The figure shows an example of pathway design based on microarray profiling of cells overexpressing a molecule previously identified to be strongly upregulated in colon adenocarcinomas, affecting PTEN signaling (yellow = upregulated; blue = downregulated).

Click on picture to enlarge

Future Research Activities 


Future studies will e.g. focus on the functional characterization of clinical relevant genes and microRNAs dysregulated in bladder cancer, colorectal cancer and prostate cancer.   

Group Leader:

Karin Birkenkamp-Demtröder, Associate professor, MSci, PhD, kbdr@ki.au.dk