Innovative therapy, monoclonal antibodies and beyond
Milan, 22 January 2016
“LXR/LXR ligands and immune mediated control of
tumor growth”
Vincenzo Russo, MD
San Raffaele Scientific Institute
Liver X Receptor(LXR) ligand/Oxysterol
generation
Bjorkhem JCI, 2002;
Pannu et al. Mol and Cell Endocrinol. 2013
LXR ligands/oxysterols are products of cholesterol oxidation
SO3
Inhibition of
COX2, IL-1b, TNFa,
MMP9, MCP-1,
IL-6 transcription
LXR SO3
SULT2B1b
RXR
SO3
Activation of
ABCG1, ABCA1,
SREBP-1c, FASN,
transcription
Active and inactive oxysterols
Cholesterol
Ester (CE)
Oxysterols
Oxidized fatty acids
RXR LXR
ABCG1 gene
ABCG1/ABCA1 Reverse Cholesterol
Transport
LXR/LXR ligand signaling
Functions
• Regulation of cholesterol and fatty acid metabolisms
(Janowsky et al. Nature 1994)
• Modulation of immune responses
(Joseph et al. Nat Med 2003)
2,3;22,23-Diepoxysqualene
Acetyl-CoA
HMG-CoA
HMG-CoA Reductase
Mevalonic Acid
Squalene
2,3-Monoepoxysqualene
Lanosterol
Cholesterol 24(S),25-Epoxylanosterol
24(S),25-Epoxylcholesterol
LXRa
Inactivates oxysterols
by sulfurylation
SULT2B1b
Zaragozic
Acid (ZA)
Bensinger et al. Cell 2008
Cui et al. JCI 2011
iT-cell proliferation
iTh17 polarization
LXR/oxysterols and immune system
Mertk-dependent phagocytosis of
apoptotic cells
iInflammation by transrepressing
COX-2, IL-6, IL-1b, etc.
A-Gonzalez et al. Immunity 2009
Joseph et al. Nat Med 2003
i T cells Macrophages Dendritic cells
Villablanca et al. Nat Med 2010
Geyeregger et al. Blood 2009
iCCR7 chemokine receptor
iAntigen presenting functions
Adapted from York & Bensinger, JEM 2013
LXR/LXR ligands (oxysterols) and tumor
microenvironment
1. Villablanca et al. Nat Med. 2010
2. Russo V. J Leukoc Biol. 2011
3. Traversari C and Russo V. Curr Op Pharmacol. 2012
Pharmacological (ZA) and genetic blockade (SULT2B1b) of
oxysterols prolongs OS of tumor-bearing mice
RMA lymphoma LLC (lung)
AB1 mesothelioma
0 10 20 30 40 50 600
25
50
75
100
Days after tumor infusion
Surv
ival (%
)
WT
Mock
SULT2B1b
RMA lymphoma
0 20 40 60 80 1000
20
40
60
80
100
Days after tumor infusionS
urv
ival (%
)
Mock
SULT2B1b
4T1 breast tumor
Adapted from York & Bensinger, JEM 2013
LXR/LXR ligands (oxysterols) and tumor
microenvironment
1. Raccosta et al. J Exp Med. 2013
2. Raccosta et al. OncoImmunology 2013
3. Traversari et al. Eur J Immunol. 2014
LXR ligands behave as chemoattractants for bone
marrow-purified neutrophils in vitro
Raccosta et al. 2013
Pro-tumor neutrophils
Adapted from Mantovani et al. Nature Rev Immunol, 2011
Adapted from The Biology of Cancer (© Garland Science 2007)
RIP1-Tag2 pancreatic tumor model
Pro-angiogenic
neutrophils
Nozawa et al. PNAS, 2006
Hanahan D. Nature, 1985
Cholesterol hydroxylases generating oxysterols
Bjorkhem JCI, 2002
Cyp46a1 is up-regulated during tumor formation in
the pancreas of RIP1-Tag2 mice
Cyp46a1
Cholesterol
• LXR activation
• Neutrophil recruitment
Cyp46a1
actin
Normal Islets Hyperplastic islets Brain
(Ctrl)
Zaragozic acid (ZA) treatment prevents the
angiogenic switch in Rip1-Tag2 mice
Generation of RIP1-SULT2B1b transgenic mice
Rip1-Tag2 Rip1-SULT2B1b
Rip1-Tag2
Rip1-SULT2B1b Rip1-Tag2 Vs.
Pancreatic tumor
development in the
absence of active
oxysterols
Tumorigenesis in Rip1-Tag2 x Rip1-SULT2B1b mice
11 weeks
Intrinsic effects of SULT2B1b in RIP1-Tag2 x
RIP1-SULT2B1b double transgenic mice
24-Hydroxycholesterol is reduced in RIP1-Tag2 x
RIP1-SULT2B1b pancreata
LC-MS Analyses carried out by Angela Bachi and Umberto Restuccia
Adapted from The Biology of Cancer (© Garland Science 2007)
Analysis of tumor microenvironment conditions
regulating cholesterol hydroxylase expression
Hypoxia
Inducible HIF-1a, Vegf and Cyp46a1 expression
during RIP1-Tag2 tumorigenesis
6 weeks
10 weeks
In collaboration with Rosa Bernardi’s group
Hypoxia and Cyp46a1 expression in vivo
Pimo/Cyp46a1/Dapi
In vitro model to study the induction of Cyp46a1
in RIP1-Tag2 tumor: bTC3 tumor cells
bTC3 tumor cells: tumor cell line derived from insulinomas arising in RIP1-Tag2 mice
(Efrat et al. PNAS: 85,9037-9041, 1988)
HIF-1 silencing reduces expression levels of Cyp46a1 in hypoxic bTC3 cells
In vitro model to study the induction of Cyp46a1
in RIP1-Tag2 tumor: bTC3 tumor cells
bTC3 cells expressing a mutant stable form of HIF-1 (Guarnerio et al. Stem Cell Reports,
2014)
HIF-1 is recruited at Cyp46a1 promoter region of bTC3 cells
HIF-1a
Cyp46a1
Cyp46a1 expression by human pNETs
Graphical Model
Range of activity
of oxysterols
Range of activity
of chemokines
Gr1/Cyp46a1/Dapi
UNIT OF IMMUNO-BIOTHERAPY OF
MELANOMA AND SOLID TUMORS
Raffaella Fontana
Marta Moresco
Daniela Maggioni
Laura Raccosta
Matias Soncini
Gianfranca Corna
MOLMED
Catia Traversari
Claudio Bordignon
Simona Porcellini
Claudia Asperti
Elena Tiziano
PATHOLOGY UNIT
Claudio Doglioni
UNIT OF FUNCTIONAL
PROTEOMICS
IFOM
Angela Bachi
Umberto Restuccia DIVISION OF EXPERIMENTAL ONCOLOGY
Rosa Bernardi
Nadia Coltella
DEPARTMENT OF BIOSCIENCES AND
NUTRITION, KAROLINSKA INSTITUTE
Knut Steffensen
Jan-Ake Gustafsson
DIABETES RESEARCH
INSTITUTE
Lorenzo Piemonti
Valeria Sordi
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