1. The decrease of MITF, a crucial melanoma transcription
factor, does not induce marks of epithelial-mesenchymal
transition, maintains the proliferation and invasivity,
but diminish the differentiation of melanoma cells
Jiri Vachtenheim, Kateřina Vlčková, and Jiri Réda
Charles University Prague, First Faculty of Medicine, Czech Republic

2. SCHEMATIC NETWORK OF MITF IN MELANOMA

3. MITF: microphthalmia-associated transcription factor
Many functions of MITF in melanoma:
- melanocyte lineage determining transcription factor
antiapoptotic protein ensuring the surival of melanocytes and melanoma cells
- lineage „addiction oncogene“ (amplified in about 10% of melanomas)
determines the expression of all differentiation markers of melanocytes
proliferation: it is believed that high proliferation is linked to high MITF
invasion/migration: lower level is believed to be associated with melanoma invasion
- senescence: very low MITF levels are believed to induce senescence
- transcriptional activation of cell cycle blockade causing facors (p21, p16 proteins) !?
- MITF has dozens of transcriptional targets
Are MITF-negative melamoas still melanomas

4. THE „RHEOSTAT MODEL“ EXPLAINING FUNCTION OF
DIFFERENT MITF LEVELS IN MELANOMA
Different MITF levels are associated with a different phenotype in melanoma cells
According to the rheostat model, low-MITF cells should be invasive and cells that proliferate rapidly are not invasive
??? ?
apoptosis resistance to apoptosis

5. All upstream MITF regulation is eliminated - in cell lines
+DOX(doxycycline)
Lentiviral inducible decrease of MITF by shRNA based on Tet-On system Tet-On-pLKO plasmid

6. INDUCIBLY DECREASED LEVELS OF MITF DO NOT HAVE ANY PROFOUND EFFECT ON CELL PROLIFERATION IN VITRO

7. INVASIVITY IS NOT INCREASED AFTER DOWNREGULATING MITF LEVEL IN CELL LINES
Scratch (wound healing) assay:

8. PROLIFERATION OF MELANOMA CELL LINES DISPLAYING LOW OR HIGH MITF LEVELS

9. DECRESE OF MITF DOES NOT CAUSE GENERAL FEATURES OF PHENOTYPE SWITCHING TOWARDS EMT
EMT MARKERS
SC MARKERS

10. DECREASED PROLIFERATION OF INDIVIDUAL CLONES RAISED FROM SINGLE CELLS - ONLY 5 OF 15 CLONES (SHOWN) HAD SLOWERED PROLIFERATIOM

11. Scratched (wound healing0 assaz
EXAMPLE OF A CLONE WITH LOW PROLIFERATION -NO HIGHER INVASION IS OBSERVED (IN ALL LOW-PROLIFERATION CLONES)
Scratch (wound healing) assay:
Scratched (wound healing0 assaz

12. MELASTATIN, A TRANSCRIPTIONAL MITF TARGET, IS PROFOUNDLY DECREASED IN CELLS WITH INDUCIBLY DECREASED MITF (real time PCR)

13. VIABILITY OF CELL LINES AFTER THE TREATMENT IN DOXYCYCLINE TO DECREASE MITF LEVEL

14. LONG-TERM (6 weeks) CULTURES OF CELL LINES - OR + DOX
(PROLIFERATION CURVES)
Western blots confirming sustained MITF decrease in long-term cultures with or without DOX

15. IN VITRO, IN MELANOMA CELL LINES:
CONCLUSIONS
IN VITRO, IN MELANOMA CELL LINES:
Downregulation of MITF through inducible shRNA does not lead to profound reduction of proliferation. Only about one third of individual cell clones arising from single cells have diminished prolireation to various degree, in three clones profoundly, by about 90%
In native established cell lines, the MITF level does not correlate with proliferation
- No decreased invasion is observed in cell lines with decreased MITF and in clones containing low MITF (invasion rather mirrored proliferation)
- the EMT gene expression pattern was inconclusive with little typical signs of EMT, SC marker Sox2 increased in MITF-downregulated cells
viability was partially decreased only in 2-3 cell lines in low-MITF cells
maintenance of cells with DOX (i.e. in low-MITF conditions) for a long time (5 weeks) did not alter their proliferation
decrease of MITF consistently led to a sharp decrese of its downstrean targets melastation (real-time PCR) and livin (Western blot)

16. K. Vlčková J. Réda L. Ondrušová P. Horák Thank you. P. Žáková
J. Vachtenheim
Charles University Prague