The key molecules and underlying mechanisms of melanoma
metastasis remain poorly understood. Using isobaric tag for relative and
absolute quantitation (iTRAQ) proteomic screening, probing of patients’
samples, functional verification, and mechanistic validation, we identified the
important role of the WD repeat-containing protein 74 (WDR74) in melanoma
progression and metastasis.
Through gain- and loss-of-function approaches, WDR74 was
found to promote cell proliferation, apoptosis resistance, and aggressive
behavior in vitro. Moreover, WDR74 contributed to melanoma growth and
metastasis in vivo.
Mechanistically, WDR74
(i) modulates RPL5[1]
protein levels and
(ii) consequently regulates MDM2 and
(iii) insulates the ubiquitination degradation of p53
(iv) by MDM2.
Our study is the first to reveal the oncogenic role of
WDR74 in melanoma progression and the regulatory effect of WDR74 on the
RPL5–MDM2-p53 pathway. Collectively, WDR74 can serve as a candidate target for
the prevention and treatment of melanoma in the clinic.
Simply stated the posited control mechanism is shown below:
WDR74 seems thus to be something which:
1. WDR74 modulates RPL5. As Fancello et al note regarding
the import of RPL5 as a ribosome as follows:
For many years, defects in the ribosome have been
associated to cancer. Recently, somatic mutations and deletions affecting
ribosomal protein genes were identified in a few leukemias and solid tumor
types. However, systematic analysis of all 81 known ribosomal protein genes
across cancer types is lacking. We screened mutation and copy number data of
respectively 4926 and 7322 samples from 16 cancer types and identified six
altered genes (RPL5, RPL11, RPL23A, RPS5, RPS20 and RPSA). RPL5 was located at
a significant peak of heterozygous deletion or mutated in 11% of glioblastoma,
28% of melanoma and 34% of breast cancer samples.
Moreover, patients with low RPL5 expression displayed
worse overall survival in glioblastoma and in one breast cancer cohort. RPL5
knockdown in breast cancer cell lines enhanced G2/M cell cycle progression and
accelerated tumor progression in a xenograft mouse model.
Interestingly, our data suggest that the tumor suppressor
role of RPL5 is not only mediated by its known function as TP53 or c-MYC
regulator. In conclusion, RPL5 heterozygous inactivation occurs at high
incidence (11-34%) in multiple tumor types, currently representing the most
common somatic ribosomal protein defect in cancer, and we demonstrate a tumor
suppressor role for RPL5 in breast cancer
2. Then that regulates MDM2[2].
As NCBI notes:
This gene encodes a nuclear-localized E3 ubiquitin
ligase. The encoded protein can promote tumor formation by targeting tumor
suppressor proteins, such as p53, for proteasomal degradation. This gene is
itself transcriptionally-regulated by p53. Overexpression or amplification of
this locus is detected in a variety of different cancers.
Prior work by Macchiarulo et al stated the following
regarding this combination[3]:
Alterations of p53 signalling pathway is the most
frequent event in human cancers. About 50% of these, albeit showing wild-type
p53, have flaws in the control mechanisms of p53 levels and activity. MDM2 and
MDMX (MDM4) are the main negative regulators of p53.
The relevance of MDM2 on the regulation of p53 levels and
activity has fostered the development of strategies aimed at restoring p53
functions by blocking the physical interaction between MDM2 and p53. As a
consequence, a number of different small molecules and peptidomimetics have
been disclosed in the last decade as inhibitors of MDM2/p53 interaction.
Recent studies, however, have thrust MDMX into the
limelight as an additional chemotherapeutic target, suggesting the presence of
a more complex relationship between MDM2, MDMX and p53. In this review article,
we report key aspects of MDMX-mediated regulation of p53, recent advances in
the structural characterization of the protein, and the progress made so far in
the medicinal chemistry of MDMX ligands.
Note that MDMX is now called MDM4, to avoid confusion. The
Macchiarulo paper was published a year ago (2011) and it presented the
connection of MDM4 and loss of p53 control in a broader context of cancer
development and spread. The Gembarska paper on the other hand has focused on
melanoma. Earlier work was performed in a Doctoral Thesis in 2007 in Rotterdam,
by Meulmeester who states[4]:
The
p53 tumor suppressor gene encodes a sequence-specific transcription
factor whose activity is either disabled or attenuated in the vast majority of
human cancers. Its inactivation occurs in about 50% of human tumors through
mutations affecting the p53 locus directly.
p53 transcriptionally activates a vast, constantly
growing number of target genes, resulting in various biological outcomes such
as cell-cycle arrest and apoptosis. Several types of stress, such as oncogene
activation, hypoxia and DNA damage, result in an increase in p53 levels and the
subsequent activation of p53 target genes (Vogelstein et al., 2000). One of the
best-characterized target genes of p53 is the mdm2 gene, which contains two
promoters.
The first promoter (P1) drives mdm2 expression
constitutively (Jones et al., 1996), while p53 binds two adjacent
p53-responsive elements within the second promoter (P2), thereby promoting
transcription of the mdm2 gene. Under normal circumstances, p53 is tightly
regulated through the interaction with its negative regulator Mdm2, which counteracts
p53 function in a number of ways.
The autoregulatory negative feedback loop, whereby p53
induces Mdm2 expression resulting in the repression of p53 function, most
probably serves as an important mechanism to restrain p53 activity in normal
cells. Therefore, uncontrolled, high expression of Mdm2 may result in improper
inactivation of p53 function. It has been shown that in 5-10% of all human
tumors Mdm2 is overexpressed, due to gene amplification, transcriptional- or
posttranscriptional mechanisms. In most of these cases the p53 gene is wild
type, presumably because Mdm2 overexpression alleviates the selective pressure
for direct mutational inactivation of the p53 gene.
As regards to the pathway discussion we presented above
Meulmeester remarks:
The complex web of ATM-mediated activation of the p53
pathway. ATM mediates direct and indirect phosphorylation of p53, while 14-3-3
binding to p53 is augmented by ATM-mediated de-phosphorylation of p53.
Phosphorylation of Strap by ATM results in the recruitment of Strap/p300
complexes towards p53 that elevates its acetylation.
A safeguard mechanism exists to ensure proper p53
activation by inhibiting its inhibitors Mdm2 and Mdmx. Phosphorylation of
Mdmx/Mdm2 attenuates their interaction with the ubiquitin protease HAUSP,
resulting in the instability of Mdmx and Mdm2. Thus ATM activates p53 via a
sophisticated mechanism, while it ensures proper activation by inhibition of
its negative regulators.
3. Which blocks p53[5].
As NCBI has noted as to this now classic gene:
This gene encodes a tumor suppressor protein containing
transcriptional activation, DNA binding, and oligomerization domains. The
encoded protein responds to diverse cellular stresses to regulate expression of
target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA
repair, or changes in metabolism. Mutations in this gene are associated with a
variety of human cancers, including hereditary cancers such as Li-Fraumeni
syndrome. Alternative splicing of this gene and the use of alternate promoters
result in multiple transcript variants and isoforms. Additional isoforms have
also been shown to result from the use of alternate translation initiation codons
from identical transcript variants.
We demonstrate this in the figure below. The complexity of
the control paths is of interest.
The figure below is another view of these control elements.
Finally below we see the interaction at the cell surfaces.
WDR74[6]
also called WD repeat domain 74. As Liu et al note:
Smads are critical intracellular signal transducers for
transforming growth factor-β (TGF-β) in mammalian cells. In this study, we have
identified WD repeat-containing protein 74 (WDR74) as a novel transcriptional
coactivator for Smads in the canonical TGF-β signaling pathway. Through direct
interactions with Smad proteins, WDR74 enhances TGF-β-mediated phosphorylation
and nuclear accumulation of Smad2 and Smad3. Consequently, WDR74 enables
stronger transcriptional responses and more robust TGF-β-induced physiological
responses. Our findings have elucidated a critical role of WDR74 in regulating
TGF-β signaling.
We demonstrate the control elements as below.
From Medical Life Sciences News[7]:
During the research, the artificially gained WDR74
function brought about a high activity in cancer cells. However, when the
function had been dropped cells failed to metastasize becoming more vulnerable
to chemotherapy. Related articles are published in Cancer Letters and Oncogene.
Except for brain cancer and some forms of blood cancer,
not the main tumor but its metastases kill the patient taking over vital
organs.
Metastases form at a certain stage of the primary tumor
progression when its cells start separating and entering the bloodstream. Such
cells are called circulating tumor cells, and they give rise to metastases
which are secondary tumors appearing in different parts of the human body.
Fortunately, just subtle minority, tenths or even
hundredths of a percent, of circulating tumor cells is capable of
metastasizing. A few years ago, Chinese scientists from the laboratory of Dr.
Lee Jia (Fuzhou University) wondered what discriminates "successful"
circulating tumor cells from "unsuccessful" ones. Searching for a
possible answer, they analyzed tumor cells (proteomic analysis) and spotted
proteins highly expressed in active metastatic cells and lost in passive ones.
One of these proteins was WDR74; its expression level in "successful"
circulating tumor cells was two times higher than in the initial tumor.
Scientists set up hypotheses stated this protein is a trigger helping a
circulating tumor cell turn into a secondary tumor.
The scientist explained that WDR74 has at least two
mechanisms of action. In different tumors, they have different priorities. In
lung cancer cells, the protein primarily regulates WNT signaling pathways,
which are active in tumor cells and passive in healthy cells of our body. In
melanoma, WDR74 indirectly affects the expression of a number of other
proteins, including the famous p53. The sequence is as follows: WDR74 regulates
the amount of ribosomal protein RLP5, which has additional, extraribosomal
properties; RLP5 regulates MDM2 protein ligase, and MDM2, in turn, leads to the
degradation of p53 protein. The question of which mechanism is responsible for
the expression of WDR74 itself remains unsolved.
Lung cancer is notorious for the lack of effective
therapy methods. The same is melanoma: the mechanisms of its progression
understood poorly. The published studies open up new paths to the development
of effective curing methods for metastases of these two cancer types with
targeted drugs. Such remedies should hit specific protein targets in the
circulating tumor cells. The drugs development is the task of the next stage of
the work of scientists from Russia, China, and Switzerland or other research
groups.
As Ruhana et al have noted in examining urinary bladder
cancer (UBC):
Non-coding mutations were common across all stages and
grades of UBC. The frequencies were: GPR126 53.0%, PLEKHS1 38.7%, TBC1D12
25.5%, LEPROTL1 23.8% and WDR74 17.2%. There was an average of 1.6 mutations per
UBC, and 74% of UBCs harboured at least one mutation. They frequently co-occur,
and commonly accompany an APOBEC mutational signature. The mutations are not
strongly associated with clinical parameters and are, most likely, early events
in the development of UBC. Mutations at these 5 non-coding hotspots are common
in UBC. Due to their high frequency across stages and grades of disease, they
should be included in UBC diagnostic biomarker panels
This may or may not be a significant finding but it does add
to the overall understanding.
References
1. Fancello et al, The ribosomal protein gene RPL5 is a
haploinsufficient tumor suppressor in multiple cancer types, Oncotarget, 2017,
Vol. 8, (No. 9), pp: 14462-14478
2. Gembarska, MDM4 is a key therapeutic target in cutaneous
melanoma, Nature Medicine, 2012
3. Li et al, WDR74 modulates melanoma tumorigenesis and metastasis
through the RPL5–MDM2–p53 pathway, Oncogene, January 2020. https://www.nature.com/articles/s41388-020-1179-6
4. Liu et al, WDR74 Functions as a Novel Coactivator in TGF-β
Signaling, J Genet Genomics, 45 (12), 639-650 2018 Dec 20
5. Macchiarulo et al, Expanding the horizon of chemotherapeutic
targets: From MDM2 to MDMX (MDM4), Med. Chem. Commun., 2011, 2, 455
6. Meulmeester , Regulation of Mdmx and its role in the p53 pathway,
PhD Univ Leiden, 2006
7. Moll and Petrenko, The MDM2-p53 Interaction, Molecular Cancer
Research, Vol. 1, 1001–1008, December 2003
8. Ruhana et al, Non-coding mutations in urothelial bladder cancer,
Bladder Cancer 5 (2019) 263–272
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