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Cellular Regulation and Control

Lecture 6 – January 27, 2022
BCH4125 – University of Ottawa
© 2022, John Pezacki.

Twitter: @PezackiLab
Email: John.Pezacki@uOttawa.ca
uOttawa.ca
Web page: http://mysite.science.uottawa.ca/jpezacki/
Figure 1.
With immunoprecipitation, Western blots, genetic reporters and
fluorescence microscopy – it was shown that the transcription
factors from the Hippo signaling pathway YAP/TAZ are associated
with the Wnt destruction complex, and translocate to the nuclei
of cells when key components of Wnt destruction complex are
missing 2
Figure 2.
With immunoprecipitation, Western blots, genetic reporters,
siRNAs and fluorescence microscopy – it was shown that
Wnt signaling causes dissociation of YAP/TAZ from the Wnt
destruction complex, and association of co-receptor LRP6
with Axin1 (stimulated by Wnt ligand) is synchronous with
dissociation and translocation of YAP/TAZ to the nucleus.
Reporter shows they can turn on Hippo signaling. 3
Figure 4.
With immunoprecipitation, Western blots, genetic reporters,
siRNAs and fluorescence microscopy – it was shown that
YAP/TAZ recruite ubiquitin ligase (β-TrCP) to the Wnt
destruction complex, and when YAP/TAZ are knocked down
with siRNAs this causes unphosphorylated β-Catenin to
translocate to the nucleus.
4
5
6
Azzolin et al. show a double role for YAP/TAZ :

1) in the absence of Wnt ligands, YAP/TAZ are part of the

destruction complex and recruit β-TrCP, thereby acting as
negative regulators of Wnt signaling.
2) in an activated pathway state, YAP/TAZ and bound β-TrCP
are displaced from Axin1 by LRP during sequestration of the
destruction complex. Free YAP/TAZ can subsequently act as
positive transcriptional effectors of Wnt signaling. The dual
role of YAP/TAZ in the Wnt pathway underlines the close
connection of Wnt and Hippo signaling.

7
 Wnt signaling in cancer

T Zhan, N Rindtorff and M Boutros Oncogene 2017, 36, 1461–1473

 Wnt signaling in cancer

T Zhan, N Rindtorff and M Boutros Oncogene 2017, 36, 1461–1473

 Wnt signaling in cancer
The impact of Wnt signaling on carcinogenesis of
colorectal cancer is well-studied

Loss of APC function is the main driver of Wnt signaling

in colorectal cancer

By genome editing of APC using the CRISPR/Cas9

technology, the carcinogenesis of CRC could be
modeled ex vivo in human intestinal organoids.

T Zhan, N Rindtorff and M Boutros Oncogene 2017, 36, 1461–1473

 Wnt signaling in cancer

T Zhan, N Rindtorff and M Boutros Oncogene 2017, 36, 1461–1473

 Wnt signaling in cancer

T Zhan, N Rindtorff and M Boutros Oncogene 2017, 36, 1461–1473

Other important growth factors
that we will discuss

VEGF and TNF-α

The basics…
 Angiogenesis

Angiogenesis is the physiological process

by which blood vessels develop (growth
factor-dependent).

During early development vessels form

through vasculogenesis, after which Judah Folkman
angiogenesis is responsible for most, if (1933-2008)
not all, blood vessel growth during
development and in disease.
In 1971, Folkman reported that solid tumor growth is
angiogenesis-dependent. He hypothesized that there was an
unknown "factor" that tumors secreted to help increase blood
supply, and that if that factor could be blocked, tumors would
wither and die. Identified fibroblast growth factor, helped to
uncover angiotensin and VEGF.
 Angiogenesis

Different factors involved that promote

proliferation & differentiation of
endothelial cells, smooth muscle cells,
and fibroblasts, recruit smooth muscle
cells, extracellular matrix production
and modification, recruitment of
lymphocytes to sites of inflammation,
formation of arteries or veins, stabilizes
junctions and nearby vessels, regulate
angioblast differentiation and
endothelial transdifferentiation,
adhesion, migration, proliferation and
apoptosis.
 VEGF signaling

Vascular endothelial growth factor (VEGF)

There are 5 main VEGF proteins:

VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and
VEGF-D.

Major actions include: stimulation of migration of endothelial

cells, mitosis of endothelial cells, matrix metalloproteinase
activity, creation of blood vessel lumen, creates new junctions
(fenestrations), inflammation and vasodilation.
 VEGF signaling

VEGFs bind with high affinity to the receptor tyrosine kinases

(RTKs) VEGFR1–R3, and VEGFR2 is the main signalling
VEGFR in blood vascular endothelial cells.

VEGFs also bind with high affinity to the neuropilin (NRP)

family members NRP1 and NRP2 and to heparan sulfate
proteoglycans (HSPGs); these are denoted VEGF co-
receptors.

The ability of VEGFs to simultaneously bind to various types of

transmembrane proteins initiates formation of multiprotein
complexes that include, in addition to receptors and co-
receptors, several non VEGF-binding auxiliary proteins, such
as integrins and ephrin B2.
 VEGF signaling

Simons, M., Gordon, E., Claesson-Welsh, L. Nat. Rev. Cell Mol. Biol. 2016, 17, 611.
 VEGF signaling

Simons, M., Gordon, E., Claesson-Welsh, L. Nat. Rev. Cell Mol. Biol. 2016, 17, 611.
 VEGF signaling

Simons, M., Gordon, E., Claesson-Welsh, L. Nat. Rev. Cell Mol. Biol. 2016, 17, 611.
 Tumor necrosis factor alpha
TNFα is a signaling protein that is
involved in systemic inflammation
and is a primary regulator of cells
of the immune system.

TNFα is a pyrogen, inducing fever Dr. Lloyd J. Old

and apoptotic cell death. It is a (1933-2011)
cytotoxic factor. Sloan-Kettering

It was identified as being secreted as well as

from macrophages, and other cell William Bradley Coley
types. (1862-1936)
UC Irvine
Produced as a transmembrane &
protein; protease cleavage yields Dr. Nancy H. Ruddle
soluble mature version of protein. Yale University
 Tumor necrosis factor alpha
Two receptors, TNFR1 and TNFR2.

These receptors are associated with a protein complex that

includes the adaptor protein TRADD.

- Induction of caspase-mediated cell death

- Activation of NF-KB transcription factor involved in

inflammatory response and cell regulation

- Activation of MAPK pathways (immediate-early response

to stress)
 Tumor necrosis factor alpha

George D. Kalliolias and Lionel B. Ivashkiv Nat. Rev. Rheumat. 2016, 12, 49.
 Tumor necrosis factor alpha

George D. Kalliolias and Lionel B. Ivashkiv Nat. Rev. Rheumat. 2016, 12, 49.
 Tumor necrosis factor alpha

George D. Kalliolias and Lionel B. Ivashkiv Nat. Rev. Rheumat. 2016, 12, 49.
George D. Kalliolias and Lionel B. Ivashkiv Nat. Rev. Rheumat. 2016, 12, 49.
George D. Kalliolias and Lionel B. Ivashkiv Nat. Rev. Rheumat. 2016, 12, 49.