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

SCHEMATIC NETWORK OF MITF IN MELANOMA

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

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

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

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

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

PROLIFERATION OF MELANOMA CELL LINES DISPLAYING LOW OR HIGH MITF LEVELS

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

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

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

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

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

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

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)

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