Discussion
FCCTX comprises a group of HNPCC families with a higher risk of
developing CRC and other associated cancers, but whose genetic basis is
still unknown. Some of the genes that have been associated with FCCTX in
recent studies include RPS20 (Nieminen et al., 2014),BRCA2 (Garre et al., 2015) and other fanconi anemia genes such asFAN1 or BRIP1 (Esteban-Jurado et al., 2016; Seguí et al.,
2015; Smith et al., 2013), BMPR1A (Evans et al., 2018),SEMA4 (Schulz et al., 2014), OGG1 (Garre et al., 2011) andSETD6 (Martín-Morales et al., 2017). However most cases remain
unexplained (Valle, 2017; Zetner & Bisgaard, 2017). With the aim of
identifying new genes involved in the cancer predisposition of FCCTX,
the whole exome was sequenced in 2 or 3 members of 13 FCCTX families. A
thorough filtering and prioritization of the identified variants allowed
a final selection of candidate variants for each family.
Here we describe the most promising candidate variant for family cc765,
a novel frameshift mutation in the PTPRT gene: c.4090dup
p.(Asp1364GlyfsTer24). PTPRT encodes a tyrosine phosphatase that
has been proven to behave as a tumor suppressor that is involved in
relevant pathways, such as the PI3K-Akt and the IL6-JAK-STAT3 pathway,
through which it regulates the expression of genes involved in cell
survival, apoptosis, cell proliferation, growth and migration (X. Zhang
et al., 2007; Y. Zhao et al., 2017). In fact, PTPRT somatic
inactivating alterations are frequently found in many tumors, including
CRC, and have been reported to act as driver mutations that promote
tumor development and progression (L.-E. Wang et al., 2013). Based on
the relevance of the gene and the effect of the mutation, this variant
was selected for further characterization.
PTPRT D1364Gfs*24 showed a compatible cosegregation with the
disease within the family, given that it was carried by 3 members
affected with different cancers while it was not present in two elderly
cancer-free relatives aged 91 and 85. However, it was also absent in a
relative diagnosed with CRC at the age of 85, but taking into account
the elevated age of onset in this patient and the prevalence of CRC in
the general population this could be perfectly explained as a phenocopy.
The cancers of which the PTPRT mutation carriers had been
diagnosed included two from the HNPCC spectrum (CRC and endometrial
cancer) and a breast cancer, which although does not belong to the
spectrum occurs with relative frequency within CRC families.
On the other hand, there was not a clear LOH of the wild-type allele in
any of the tumors tested. Nonetheless, PTPRT promoter has been
recently reported to be frequently hypermethylated in sporadic CRC and
other tumors (Laczmanska et al., 2013; Peyser et al., 2016). Indeed, the
promoter region of PTPRT was found to be hypermethylated in the
two tumors of the carriers that were tested (breast and colon tumors),
which could be a different mechanism of inactivation of the wild-type
allele in the tumors from the carriers. In fact, an allele-specific
expression assay showed that the expression of PTPRT ´s wild-type
allele was significantly reduced in the colorectal tumor when compared
to healthy colon from the same individual, while no decrease was
observed in the expression of the mutant allele. This supports that the
epigenetic silencing mainly affects the wild-type allele and could be
considered a second hit involved in the inactivation of this tumor
suppressor gene.
Regarding the effects of the variant on the protein, D1364Gfs*24 is a
frameshift mutation that affects PTPRT’s second catalytic domain, known
as D2. PTPRT D1364Gfs*24 results in the loss of the last 97 amino
acids of the protein, including 36.2% of the D2 domain and a
considerable amount of essential D2 residues. Actually, the majority
(69.2%) of residues directly surrounding the substrate’s
phospho-tyrosine are lost with this mutation (Lui et al., 2014).
Although the first phosphatase domain (D1) is the one known to be
responsible for the phosphatase activity of the protein per se ,
D2 is responsible for the regulation of this activity and has been
proven to be essential for PTPRT’s activity (Y. Zhao et al., 2017). As a
matter of fact, Wang et al. reported that just a missense mutation
affecting D2’s residue 1368, which is lost in our mutant, was enough to
decrease the enzyme’s activity by half (Z. Wang et al., 2004). The
relevance of this second catalytic domain was also pointed out by Zhang
et al., who showed how the deletion of the D2 domain had significant
effects on the levels of phosphorylated PTPRT substrates and the
expression of its downstream target genes (X. Zhang et al., 2007).
Therefore, it can be predicted that this mutation will result in
significant consequences for the activity of this phosphatase.
PTPRT is known to dephosphorylate two main target proteins, STAT3 and
paxillin (X. Zhang et al., 2007; Y. Zhao et al., 2017, 2010), both of
which are well-known oncogenes (Bromberg et al., 1999). These two
proteins are activated upon phosphorylation by different protein
tyrosine kinases, so the PTPRT-mediated removal of the phosphate group
results in their inactivation. This results, in turn, in the inhibition
of their downstream pathways, through a decreased phosphorylation of
paxillin’s target proteins (Y. Zhao et al., 2017, 2010) and a decreased
expression of STAT3’s target genes (X. Zhang et al., 2007).BCL-XL and SOCS3 are two of those STAT3’s target genes,
which have been proven to show increased expression upon PTPRT
depletion, and even upon deletion of the D2 domain (X. Zhang et al.,
2007). Consistent with the hypothesis that this germline PTPRTvariant may be involved in the cancers of this FCCTX family, the tumors
from the two carriers tested presented a significantly increased
expression of BCL-XL , which is an oncogenic driver in CRC (Scherr
et al., 2016). This was observed in both the colon and breast tumors
when compared to healthy tissue controls.
In contrast, SOCS3 expression was decreased in both tumors.
Nonetheless, SOCS proteins are negative feedback regulators of the
JAK-STAT signaling pathway (Inagaki-Ohara, Kondo, Ito, & Yoshimura,
2013; Jiang et al., 2017), and it has been proven that SOCS3 is
usually downregulated in CRC, even when IL-6 and STAT3 are
upregulated (Chu et al., 2017). This is thought to occur through
different mechanisms, such as the hypermethylation of SOCS3 gene
promoters, allowing inflammatory cytokines IL-6 to activate STAT3’s
signaling pathway while inhibiting the expression of SOCS3 (Chu
et al., 2017), with the purpose of inactivating its negative feedback.
This negative regulation of SOCS3 expression, mediated by the
activation of IL-6/STAT3, leads to imbalance and sustained activation of
STAT3 signaling pathway (Chu et al., 2017). The same study by Chu et al.
also showed that SOCS3 plays an important role inhibiting tumor
development and that reduced SOCS3 ’s expression affects
tumorigenesis and CRC progression, promoting growth and metastasis (Chu
et al., 2017). For all the things mentioned, both results are compatible
with a pathogenic role of this PTPRT variant.
As previously discussed, PTPRT ’s association with cancer has been
well studied, but this is the first time that PTPRT is linked to
hereditary cancer. Interestingly, a somatic mutation affecting the same
codon (COSV62009665) is associated with CRC according to COSMIC (Tate et
al., 2019). Even though the role of this protein in tumor development is
undeniable, more studies are needed to confirm its involvement as a
cancer susceptibility gene. Studying this gene in a larger cohort would
help with this task. As a matter of fact, the screening of PTPRTin an independent familial/early-onset CRC cohort identified one
additional rare missense germline variant located inside the D1 domain
in an early-onset CRC patient. However, the functional effect of this
variant has not been determined.
Last but not least, it should be pointed out that even though thisPTPRT mutation had the highest potential for the explanation of
the increased cancer risk, another three candidate variants were
prioritized for this family: two missense variants in MAP3K6 andABTB1 , and a splice region variant in INVS . MAP3K6 is
involved in the regulation of VEGF expression and has also been
reported to act as a tumor suppressor (Gaston et al., 2014). In
addition, germline mutations in this gene have been associated with
familial gastric cancer (Gaston et al., 2014). ABTB1 is a mediator of
the PTEN signaling pathway reported to suppress the growth of cancer
cells by the inhibition of the cell cycle (Unoki & Nakamura, 2001).
Finally, INVS acts as a molecular switch between the different Wnt
signaling pathways, inhibiting the canonical Wnt pathway (Simons et al.,
2005), and homozygous INVS mutations have been associated with
juvenile nephronophthisis (Bellavia et al., 2010). Although PTPRTD1364Gfs*24 is the best candidate variant for this family and the
results presented in this report support its causality, we cannot rule
out the possibility that these candidate variants – or even other
genetic or environmental factors – may be contributing, independently
or together, to the increased cancer susceptibility of the family or
modifying the effect of this PTPRT mutation.
Taken together, the results here presented point to a probable causal
role of the germline variant PTPRT c.4090dup
p.(Asp1364GlyfsTer24) in the cancer susceptibility of the carrier
family. For that reason, we propose PTPRT as a novel cancer
predisposition gene. However, more research is necessary to confirm the
causality, penetrance, conferred risk and preferred cancer location. The
screening of this gene in additional familial colorectal cancer cohorts
– or even in other high-risk families – will help us clarify its role
in cancer predisposition. Although PTPRT’s role in cancer initiation and
progression has been well stablished, this is the first time that aPTPRT germline variant is linked with cancer susceptibility and
hereditary cancer, which highlights the relevance of this work.