10 years after surgical resection of melanoma region on the cheek.

To be sure, BRAF mutation is a deriver event for melanoma development, but BRAF inhibitor vemurafenib does not seem to show dramatic clinical outcome.

Cancer Metabolism

Mitochondria

Mitochondrial Biogenesis

!C"
ero#isome proliferator$activated receptor$% coactivator & !C'$"( is a member of a famil) of transcription co$activators that pla)s a central role in the regulation of cellular energ) metabolism. !C" interacts with numerous transcriptional factors depending on the stimulation. PGC-1 stimulates mitochondrial biogenesis and promotes the remodeling.

PGC1 interacts with numerous transcriptional factors depending on the stimulation.

!C" is a better prognostic mar*er for patients with melanoma.

a!or "uestion
#hether and how oncogenic signals impact PGC1 e$pression and what are the metabolic and growth conse%uences this might cause to the melanoma phenotype&

#orking 'ypothesis
PGC1a could be aberrantly acti(ated in some tumors) thereby conferring them an adapti(e ad(antage.

+pecific Aim
*o demonstrate that PGC1 defines the metabolic and energetic program of human melanoma cells.

Relative e#pression level of !C" and !,+A anal)sis in human melanoma patients

There is a diversit) of !C" mR-A amount among human melanoma cell lines.

PGC1 negati(e melanoma cell line +,-. was deri(ed from a metastatic tumor produced by the PGC1-positi(e parental cell line +,-.P.

There is a diversit) of mitochondrial amount among human melanoma cell lines.

.eletion of !C" in A/01 cells leads to mimic !C"$negative melanoma cells.

+hort +ummar)"
!C"( is elevated in a subset of human melanoma tumors and derived cell lines. There is a diversit) of mitochondrial amount among human melanoma cell lines, depending on transcription of !C"(. .eletion of !C" in melanoma cells leads to mimic !C"$negative melanoma cells.

/*0) the lineage factor for melanocytes

b232$4ip transcriptional factor &' Microphthalmia$associated transcription factor &M5TF' acts as a master regulator of melanoc)te differentiation. M5TFphennot)pe -6 TF,/

M5TF is an amplified oncogene in a fraction of human melanomas.

M5TF has the same function between in normal melanoc)tes and melanoma cells777

MT89TM,C! Therapeutic +trateg)

MT8 activation of M5TF and conse:uentl) T;R activates the melanoma$specific antifolate activit) of TM,C!, leading to depletion of cellular dTT and ,<F"$mediated apoptosis.
6o$10&&&

Cancer Cell 101,213410.-115.

,#pression of !C" and M5TF is positivel) correlated each other.

'eat map of the top 10 genes differentially e$pressed between the top and the bottom 1.7 of melanoma samples ranked by PGC1 e$pression le(els.

,#pression of !C" and M5TF is positivel) correlated each other.

,#pression of M5TF is necessar) to maintain !C" and T;R in M5TF&high' melanoma cells.
/*0 m9:+ *89 m9:+

/*0;
&lineage$specific transcription factor'

PGC1) *89;

5nduction of M5TF into M5TF&negative' A/01 melanoma cells results in increased !C" and T;R e#pression.

Anti$o#idant

Mitochondria

/*0<
&lineage$specific transcription factor' 6=>14 supero$ide dismutase 1 Gp$14 glutathione pero$idase1 :dufs,4 Core subunit of the mitochondrial membrane respiratory chain :+>' dehydrogenase ?Comple$ /@ C=A.+4 cytochrome c o$idase subunit

PGC1) *89<

Ch5 anal)sis at the !C" promoter in A/01 cells using an antibod) against M5TF and immunoglobulin ! &5g!' as a control.

Chromatin immunoprecipitation &Ch5 ' assa)s showed that endogenous M5TF is bound to the !C" promoter

Chromatin immunoprecipitation &Ch5 ' assa)s showed that endogenous M5TF is bound to the !C" promoter
? /*0-high melanoma cell line@

+hort +ummar)<
,#pression of !C" and M5TF is positivel) correlated each other. !C"($positive melanoma cells e#hibit increased mitochondrial metabolism.

Melanoc)te$lineage specific transcription factor M5TF is bound to the !C" promoter.

!C"$positive cells e#hibited increased o#)gen consumption rates &=CR' and decreased gl)col)sis.

Glycolysis

FCC is a protonophore &2> ionophore' and uncoupler of o#idative phosphor)lation in mitochondria.

!C" depletion is positivel) correlated with =CR.

!C" e#pression increases glucose upta*e and decreases gl)col)sis.

shPGC1

PGC1 /nduction

.epletion of !C" in !C"$poitive melanoma cells induces apoptosis.

-positi(e -negati(e -positi(e

!C" e#pression decreases cleaved AR and caspase$/,? but not @ in A/01 and MeAo cells.
-positi(e -negati(e -positi(e

+hort +ummar)/
!C"($positive melanoma cells depend on TCA c)cle in mitochondria, whereas !C"($ negative melanoma cells depend on gl)col)sis.

!C"( activates the intrinsic mitochondrial apoptotic pathwa) and strongl) suggest that !C"($positive melanoma cells have become dependent on !C"( for survival and proliferation.

Trolo# and B$aggregation assa)

Trolo# is an anti$o#idant molecule, a water$ soluble derivative of Citamin ,.
Bow 9=6 'igh 9=6

monomer

polymer

!C" depletion increases R=+.

!C" depletion increases R=+.

!C" e#pression contributes to !+2 production, thereb) inhibiting R=+$induced apoptotic cell death.

-AC or Trolo# recues the increased susceptibilit) to o#idative stress.

!C"( enhances increases the e#pression of R=+ deto#ification genes, thereb) inhibiting apoptotic pathwa).
+nti-o$idant 9escue

+=.< protein levels are substantiall) decreased in !C"($depleted melanoma cells, and ectopic e#pression of !C"( in A/01 increases R=+ deto#ification genes

Cnockdown of PGC1-targeting molecules decreases mitochondrial metabolism and G6' production) mimicking PGC1-negati(e melanoma.

Cnockdown of PGC1-targeting molecules decreases mitochondrial metabolism and G6' production) mimicking PGC1-negati(e melanoma.

Cnockdown of PGC1-targeting molecules decreases 9=6-induced apoptotic pathway) which is rescue with :+C or *rolo$ treatment.

/*0) the lineage factor for melanocytes

Microphthalmia$associated transcription factor &M5TF' acts as a master regulator of melanoc)te differentiation. M5TF is an amplified oncogene in a fraction of human melanomas.

hosphor)lated histone 2<A8 &%2<A8' is used as a biomar*er of cellular response to double$strand$brea*&.+B'.

Clin Cancer Res <D"DE"FGH1/<$H1H<.

/mmunostaining of >>9 proteins in senescent cells. ?Protocol Exchange) 100D@

=vere#pression of !C"( without M5TF helps melanoma cells overcome .-A damage response.
=vere#pression of !C"( in A/01 cells

Treatment of M5TF or !C"( $depleted cells with the antio#idant -AC partiall) bloc*ed the apoptotic effects.

+hort +ummar)H
!C"($positive melanoma cells e#hibit increased R=+ deto#ification capacit), thereb) preventing apoptosis.

!C"($positive melanoma cells are highl) sensitive to the induction of R=+ due, at least in part, to the disruption of the mitochondria respirator) comple#.

.own$regulation of !C"( contributes to the phenot)pic effects of M5TF *noc*down in !C"($ e#pressing cells.

,5TC, h)drogen pero#idase, and piperlongumine but not B$RAF inhibitor 38HD/< increases R=+.

/ndian long pepper

piperlongumine

!C"( depletion and R=+$inducing drug such as piperlonguime show the s)nergistic effect in vivo tumor formation.

!C"( depletion increases the susceptibilit) to R=+ in activation of apoptotic pathwa), thereb) decreasing the tumor si4e b) piperlongmine.

+hort +ummar)1
!C"($negative melanoma cells are more gl)col)tic and sensitive to R=+$inducing drugs.

!C"( contributes to the tumor development and growth b) inducing anti$o#idant capacit).

Ta*e 2ome Message

'eterogeneity in cancer cell metabolism and o$idati(e stress
undifferentiated

differentiated