TWIST1

The TWIST1 gene is located on 7p21.2. It is part of the basic helix-loop-helix (bHLH)
gene family. It is a zygotic regulatory gene that activates other gene cascades to
determine orofacial and limbic foetal cells, starting from the mesoderm. Heterozygous
loss-of-function mutations at this stage can present as abnormalities in skull structure,
as part of the Saethre-Chotzen syndrome, or result in isolated cases of
craniosynostosis. In adults, Twist1 expression can be found in human peritoneal
mesothelial cells, endometrial fibroblasts and white adipocytes. 54 Twist1 has
connections to major developmental signalling pathways, including TGF-, Wnt, Notch,
and growth factor receptor signalling cascades, which are all linked somewhere to the
EMT program.
Its roles in metatasis come from its influence on angiogenesis, invadopodia,
extravasation, and chromosomal instability.55 Twist1 can be activated by STAT3 or be
induced by the inflammatory cytokine TNF via IKK- and NF-B p65 activation.
Cytokines in the tumor microenvironment can also activate Stat3 via JAK kinases to
induce Twist1 expression. In a study on sarcoma, several of Twist1s direct target genes
were identified to be markers of EMT and stemness - ID2, ABCA1, Prrx1, SNAI2,
PDGFRA, and PDGFR. These genes are also essential for mesenchymal character and
self-renewal.
During EMT development, Snail, Zeb, and Twist induce epigenetic silencing at the Ecadherin promoter through hypermethylation and histone deacetylation. The loss of Ecadherin is a key feature of the transitioning phenotype. It translates to a loss of the tight
adherens junctions joining cells, such that individual cells can easily migrate 56. Bcl-2
could assist Twist1 in translocating to the nucleus under hypoxic conditions where
Twist1 acts as transcription factor and HIF-1 can mediate Twist1 expression by binding
to the hypoxia-response element in the Twist1 proximal promoter to upregulate Twist1
expression, thus promoting metastastic EMT phenotypes.54

Through a research teams recent effort to purify the Twist1-associated protein complex,
Twist1 was found to act, sometimes indirectly, on various parts of the Mi2/nucleosome
remodeling and deacetylase protein complex (Mi2/NuRD) made up of Rb-associated
protein 46 (RbAp46), Mi2, metastasis-associated protein 2 (MTA2) and histone
deacetylase 2 (HDAC2). When Twist1 leads this gene repression protein complex to the
E-cadherin promoter, E-cadherin expression is inhibited. 57 The Mi2 Part of the
Mi2/NuRD complex contains chromatin-dependent ATPase activity and aid nucleosome
movement by means of a sliding mechanism. Together the HDAC and ATPase of the
Mi2/NuRD yield densely packed hypoacetylated nucleosomes for muting genes. 58
Although the Mi2/NuRD complex has a MTA component that specifies its distinct
function, its primary function is to repress gene expression across many areas, and can
have its role in cancer initiation and development. Upon knockdown of MTA2 or
RbAp46, E-cadherin expression resumes in cancer cells, while inhibiting their migration,
invasion and metastasis, just as knockdown of Twist1 does. These findings not only
established mechanistic and functional links between Twist1 and the Mi2/NuRD
complex but also clarified the roles of various Mi2/NuRD complex components in EMT
induction.54

Another differential change in EMT is invadopodia formation. Invadopodia are

specialized cellular structures that promote matrix degradation and invasion This
happens from the activation of PDGFR and Src. TGF- was also shown to induce
invadopodia formation via upregulation of Twist 1 in EpH4 and MCF10A mammary
epithelial cells, as well as by over-expressing focal adhesion protein Hic-5. Expression
of proteases can further infuce EMT by breaking down cell-cell junctions and forming
feedback loop to exacerbate malignant transformation.
The binding of Twist1 to p53 prevents apoptosis and allows its cooperation with other
oncogenes such as Her2 and H-ras to promote malignant transformation, evincing its
roles in tumor initiation.5 Twist1 effects appears to affect carcinomas and sarcomas
differently where Twist1 promotes tumor invasion but represses self-renewal of
carcinoma but Twist1 facilitated both invasion and self-renewal of synovial sarcoma in
our study.
N-cadherin and vimentin are meanwhile overexpressed. This happens as Twist1 also
upregulates mesenchymal markers expression. Nuclear localization and accumulation
of Twist, along with the expression of its target gene N-cadherin, is mediated by and
dependent on 1 integrin signaling.59

VERSICAN
VCAN is located on 5q14.3. Its transcription is mediated by -catenin, a central
molecule in wnt pathway. Versican, its product, is part of the extracellular matrix
chondroitin sulfate proteoglycan family. Versican is expressed in the epithelial cells of
skin and nervous system cells. There are five possible types of Versican: V0, V1, V2, V3
and V4.

Versican has high presence in areas of active mitosis and migration, such as during
embryonic development. The protein is crucial to the formation of the heart and guiding
neural crest cell migration. In adults, they are present in loose connective tissues close
to the elastic fibre network including cartilage and basal layer of epidermis. Besides
ensuring the integrity of the extracellular matrix with hyaluronan, it also helps maintain
the structure and consistency of the vitreous fluid in the eyeball and is part of the
interstitial fluid. It participates in cell adhesion, proliferation, migration and angiogenesis
via various actions on its domains. Its GAG chains are negatively charged and help it
attract water, thus the viscoelasticity of the pericellular microenvironment. 60
V4 is found and upregulated in human breast cancer. The 4 Versican variants also differ
in the number of attached chondroitin sulfate (CS) chains. It is observed in tumour
tissue that the chondroitin sulfate chains tend to be oversulfated, which may control
ligand interaction and thus cancer progression. V2 is found to increase angiogenesis for
endothelial cells by mitosis retardation and boosting fibronectin synthesis. Another study
reveals the capacity of V3 to promote a specific phenotype where ASMCs are
transduced with V3.60
VCAN modulates cell proliferation, adhesion and migration during development and
wound healing cell. In neural cells, versican is crucial to axonal guidance. There is
evidence from studies that demonstrate upregulation of versican along with other
hyalectins following central nervous system injury. Meanwhile in inflammatory
processes, Versicans ability to act on immune cell receptors and chemokines can be
significant in metastasis. A study showed that the high levels of versican in Lewis lung
carcinoma affect TLR2 to stimulate the release of inflammatory cytokines including
tumor necrosis factor- (TNF) and IL-16. The litigation of immune receptors by
versican contributes to activation of multiple cell types and activating cytokines in many
pathological conditions, and promote tumor expansion and metastasis.
Versican works together with several extracellular matrix components near cell surface
to produce a mechanically active biopolymer between cell that effects the cells ability to

shape shift, adhere, proliferate, migrate and assemble other ECM proteins and survive.
These participating ECM components include HA, tenascin-R and -C, thrombospondin
1, fibronectin, and fibrillin. Versican and these molecules that bind to it, through
changing the mechanical stiffness around cells, can cause changes to
mechanotransduction influencing cell behavior and phenotype. Versican further
establishes fine control over cell activity, behavior and the induction of metastasis by
selective releasing cytokines and growth factors from its reservoir. For example, T cell
does not just adhere, but will barely be able to migrate or produce IL-10 when in contact
with a versican-enriched ECM. This exemplifies the impact of Versican over the
phenotype of immune cells.60

WNT11
Wnt genes code for extracellular signaling factors. The Wnt signaling occurs either
through the canonical -catenin-dependent Wnt pathway or non-canonical -catenin
independent Wnt pathway. The former control cellular proliferation, stem cell
maintenance and somatic cell reprogramming while the latter, including Wnt/Planar cell
polarity (PCP) and the Wnt/calcium (Ca2 +) pathway prepares the body axis during
embryogenesis, as shown in mice.61 Wnt-11 is expressed in cardiac tissue,
gastrointestinal tract and the red pulp of spleen. It is found to activate cardiogenesis.
62

Mutations are found to contribute to kidney defects. Ulrich et al have reported that

Wnt-11 controls tissue morphogenesis by modulating cell cohesion mediated by Ecadherin.63

In a study on nephrolithiasis, a condition of renal fibrosis and stones, Wnt signaling
pathways were said to be linked to TGF-1, an activator to its components, and to EMT.
In primary and immortalized renal epithelial cells, Wnt11 is directly regulated by TGF-1
through Smad3. Conversely, Wnt11 also increases expression of TGF-1 and is
required for stimulating high mesenchymal gene expression, such as Zeb1 and Snail1. 64

In the non-canonical pathway, Wnt11 is a direct transcriptional target of the ERR/-cat

complex that affects E-cadherin expression, whose low levels facilitating a promigratory
phenotype change. There is evidence for an autocrine regulatory loop for this. 65 Most
studies show Wnt11 activating the non-canonical Wnt signaling pathways through the cJun N-terminal kinase (JNK) or calcium/calmodulin-dependent protein kinase II
(CAMKII).61 In the CamKII or Wnt/Ca2+ pathway, the cell experiences G protein
dependent increases in intracellular calcium and then activation of protein kinase
C(PKC), shown to increase intestinal epithelial cellular migration. Also, both CAMKII and
PKC stimulate actin cytoskeleton rearrangements, critical for cellular migration, and
facilitating cell differentiation to mesenchymal phenotype. WNT11 has been shown
recently to also act via canonical Wnt pathway. 66

The precise function of wnt11, however, varies in different cell types. Its effects are
context dependent and sometimes contradictory. Wnt11 was shown to increase the tight
and gap junctions in a quail mesodermal cell line QCE6 as part of the cardiomyocyte
differentiation process. In contrast, Wnt11 in vitro effected E-cadherin clearance in a rat
intestinal epithelial cell line IEC6, inspiring proliferation and migration (48). These
examples elucidate the importance of the cellular context to the expression of wnt11. 67