| Abstract |
Tpl2 and Gfi1 genes are considerably new members of the cell oncogene family for
which we speculate a role in human cancer development as it is already understood that they
contribute in the development of lymphoid neoplasms of T-cell origin mostly in animal
experimental models.
Specifically, Tpl2 kinase (or MAP3K8) induces T-lymphoblastic lymphoma growth
when expressed in transgenic mice rearranged (truncated) at its C-terminus, while
overexpression of the wild type protein is also oncogenic. In humans it has been implicated in
Hodgkin’s lymphoma, in nasopharyngeal cancer and breast cancer. Recently a 3’ end
mutation was discovered in lung cancer. Its normal function includes participation in Tlymphocyte
activation and IL-2 production. It is inhibitory for the cell-cycle regulatory
protein p27kip and induces various transcription factors that stimulate cytokine production like
IL-2 and promote cell proliferation. Moreover it participates in TNF-α production from
macrophages as well as TNF-α and CD40 signaling in macrophages and B-lymphocytes. The
truncated kinase seems to be more potent.
Accordingly, the gene that encodes for Gfi1 transcription factor is also a proviral
integration site in MoMuLV-induced T-cell lymphomas and confers a partial IL-2
independence in the cell lines derived from these lymphomas. Transgenic mice develop Tcell
lymphomas in a low rate which can be substantially increased by the simultaneous
expression of c-Myc or Pim. It is also overexpressed in B-cell lymphomas induced by
MoMuLV in Emu-myc mice. Its oncogenic potential is associated with progression rather
than initiation of the process. It also plays an important role in hematopoiesis regulation.
ERK pathway is important for Gfi1 regulation, possibly through the transcription factor AP-1
following TCR activation. Up to date there has been no study in humans addressing its role in
hematologic neoplasms and no correlation with the Tpl2 gene which is a ΜΕΚ/ΕRΚ/ΑΡ-1
pathway mediator.
Finally, Pim1 kinase belongs, like MAP3K8, in the cytoplasmic serine/threonine
kinases and possesses anti-apoptotic and cell proliferation induction properties. Its oncogenic
capacity has been confirmed in experimental models as well as in humans, where it has been
implicated in B-cell lymphomas, prostate cancer and myeloblastic leukemias. It is important
for the manifestation of the anti-apoptotic properties of various cytokines, especially those
that activate STAT3. It is considered an oncogene with a role in the initiation of the
malignant transformation during which it can co-operate with other oncogenes. Mikkers at al.
used insertional mutagenesis with MoMuLV in ΕμMyc+/Pim1- mice in order to analyse
proviral integration sites and they discovered 9 sites that are associated with mutual
exclusion, in other words not co-current integration with Pim1. One of them was the Tpl2
gene which means that concerning oncogenesis Tpl2 should act parallel or downstream of
Pim1.
In humans the B-cell malignancies are far more frequent than the T-cell ones, at least
in the Western world. Thus, in the present study we included a series of patients with T- and
B-cell lymphoid neoplasms (leukemias or lymphomas with a leukemic presentation) aiming
in studying: a) Tpl2, Gfi1 and Pim1 expression using semi-quantitative conventional RT-PCR
as well as SybrGreen real-time qPCR in peripheral blood lymphomonocytes to explore the
possibility of overexpression and to reveal co-operative or mutual exclusion relations b) the
possibility of gene amplification of Tpl2 with multiplex PCR based on the results by
Sourvinos at al. on human breast cancer and c) TNF-α and IL-2 serum levels by ELISA
technique as well as IL-12 and INF-γ levels which are associated with diseases like the LGLleukemias
and B-CLL.
We selected 24 patients from the Hematology clinic of the University Hospital of
Heraclion. Twelve suffered from B- and 12 from T-cell lymphoid malignancies. We also
included a patient with B-cell lymphocytosis which proved to be of reactive type due to
rheumatoid arthritis and 22 healthy volunteers. 18 ml of peripheral blood was drawn from
each patient. RNA and DNA were isolated from the peripheral blood mononuclear fraction
which was extracted by the ficol-gradient centrifugation method.
Conventional RT- PCR results displayed a low dynamic range in the estimation of
relative gene expression, although no different from real-time qPCR in distinguishing
samples with overexpression.
Among patients with T-cell lymphoid malignancies 33% had overexpression of Tpl2
up to 5.2 times of mean control expression and all of these were patients with LGL- chronic
lymphocytic leukemias. Three of them had co-existent immune neutropenia. The same
patients displayed Gfi1 overexpression. Pim1 overexpression was found in 6 out of 12
patients, specifically the T-LGL group plus 3 patients with aggressive T-cell malignancies.
Among patients with B-cell lymphoid malignancies 9 (75%) showed Tpl2
overexpression. These were all the patients with B-CLL plus one patient with splenic
lymphoma of marginal cell type, one patient with mantle cell lymphoma and marginally one
patient with B-CLL/Richter’s transformation. None had Gfi1 overexpression. Moreover, 9
patients had Pim1 overexpression: the CLL patients and patients with mantle cell lymphoma,
as well as the patient with lymphoma of marginal cell type.
Two-tailed Spearman’s rank order correlation analysis showed interesting relations
between the 3 genes: a statistically significant positive correlation of the expression of all
three of them in T-cell malignancies and a Tpl2 - Pim1 expression positive correlation in Bcell
malignancies. In each case there was no negative correlation between them.
Tpl2 gene load analysis by multiplex PCR in genomic DNA showed no gene
amplification in any sample.
Finally, evaluation of IL-2, INF-γ, TNF-α and IL-12 serum levels and correlation
with Tpl2NORM (e.g. Tpl2 expression) showed the following results: IL-2 was uncountable in
all samples. Patients with T-cell lymphoid malignancies generally and specifically with LGL
leukemias had statistically higher TNF-α levels than the control samples, without a
statistically significant correlation between Tpl2NORM and TNF-α. They also had higher levels
of IL-12 with a positive correlation between Tpl2NORM and IL-12. INF-γ levels were no
different between patients and controls, although in the LGL group there were single cases
with very high values. Likewise, patients with B-cell lymphoid malignancies had higher
TNF-α than the controls without a statistically significant correlation between TNF-α and
Tpl2NORM. Among those, patients with mantle cell lymphoma had a trend towards a higher
TNF-α level than the rest. Moreover, patients with B-cell lymphoid malignancies had as a
whole higher IL-12 and INF-γ levels compared to the controls with no distinguishable group.
There was a positive correlation between Tpl2 expression and IL-12 levels.
In summary, we showed that chronic leukemias of the LGL type show
overexpression of Tpl2, Gfi1 and Pim1. Thus, the above mentioned mutual exclusion
relationship between Tpl2 and Pim1 is not proven, while we confirm the co-operative
relationship of Pim1 and Gfi1. Furthermore, Tpl2 mRNA overexpression is not due to gene
amplification contrary to the corresponding breast cancer study, which is quite expectable,
since this is a rare phenomenon in lymphoid neoplasms. Pathogenesis of the LGL
malignancies is not very well understood. They are indolent neoplasms of the CD3 positive
T/NK-like lymphocytes usually with a phenotype CD8+, CD4–, TCR αβ+, CD16+, CD57+
or of the NK cells with CD3-, TCR- , CD2+, CD16+, CD56+ phenotype. The genes in our
study function in many levels that could promote oncogenesis in these neoplasms. Thus, Tpl2
and Pim1 induce IL-2 production, while the same genes as well as Gfi1 contribute in IL-2
induced T-lymphocyte activation. Tpl2 may be implicated though JNK activation in the Tcell
differentiation towards CD8+ following TNF-α stimulation, as it was shown in the
experimental model of inflammatory bowel disease in the ΤnfΔARE mice. High levels of IL-12
in these patients were positively correlated with Tpl2 expression which is a known p38
pathway activator, while the same patients had also high TNF-α levels. Thus, neutropenia in
these patients could be a result of the increased TNF-α production by the malignant cells,
resulting from IL-12 and IL-2 stimulation and mediated by p38 and ERK pathway activation
by Tpl2 respectively.
In the most aggressive malignancies of T-cells, like T-cell prolymphocytic leukemia,
peripheral T-cell lymphoma and Sezary syndrome in progression we only found
overexpression of Pim1, which apparently acts in concert with other genes in the malignant
process.
In the B-cell lymphoid malignancies that we studied we showed increased Tpl2
expression in patients with B-CLL and in two patients with splenic lymphoma of marginal
cell type and mantle cell lymphoma respectively. In the same patients we observed Pim1
overexpression. Pim1 contribution to human lymphoid malignancies, especially B-cell
lymphomas is well known. Molecular biology of B-CLL is a provocative issue, since it is still
not thoroughly understood. Many cytokines have anti-apoptotic effect in CLL cells and can
also be produced by them. Moreover, NF-κΒ overexpression has a critical role in CLL cell
survival, while inhibition of MAPK pathway MKK3/6/p38 shortens their survival in vitro.
ZAP70 kinase expression, which confers a bad prognosis, is induced though Ras/MAPK in
the normal T lymphocytes, although we don’t know the mechanism of its activation in the
CLL cells. All of the above offer us a possible mechanism of action for the Tpl2 kinase in
CLL. Increased levels of TNF-α, INF-γ and IL-12 have an anti-apoptotic effect. Tpl2 can
induce NF-κΒ activation by various cytokines or by CD40 activation. At the same time Tpl2
along with Pim1 can promote IL-2 production by the leukemic cell, thus creating a positive
feedback circuit in cytokine production and activity.
Gfi1 was not induced in B-cell malignancies, apart from a case of splenic lymphoma,
which is in accordance with experimental data.
Finally, mantle cell lymphoma, in which we basically found Pim1 and in a lesser
degree Tpl2 overexpression, is a well studied disease which is characterized by cyclin D1
overexpression. Overexpression of NF-κΒ has also been shown in this aggressive lymphoma,
while an important step in disease progression is the inhibition of the G1 regulatory proteins
p27kip and p21 waf1. There is already data for a Pim1 role in the blastoid variant of the disease,
while Tpl2, with its aforementioned activities (IL-2 production, NF-κΒ induction) and its
inhibitory result on p27kip could play a combinational role in this lymphoma.
In conclusion, Tpl2 and Gfi1 overexpression can contribute in the development of
some human T, B and NK lymphoid neoplasms, in collaboration with other oncogenes like
Pim1, thus confirming experimental results in animal models and cell cultures concerning
their role in lymphoid malignancies.
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