Part II: Increased serum placenta growth factor level is significantly associated
IV. Discussion
Part I: Increased placenta growth factor mRNA level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
In this study, we measured the PlGF mRNA level in 63 paired OSCC and non-OSCC tissue samples.
The relatively lower mean PlGF mRNA CT value in OSCC than in non-OSCC tissues indicated an
increased PlGF mRNA expression in OSCC tissues than in non-OSCC tissues. The finding of PlGF
mRNA expression in normal-looking oral mucosal (non-OSCC) tissue adjacent to OSCC also
suggests that the PlGF mRNA expression is an early event in oral carcinogenesis. Takahashi et al.
(1994) found a significantly higher PlGF mRNA expression in hypervascular renal cell carcinoma
tissues than in adjacent normal kidney tissues. Chen et al. (2004) reported a significantly higher
PlGF protein level in gastric cancer tissues than in the corresponding non-cancerous mucosal tissues.
Parr et al. (2005) also demonstrated that the PlGF protein expression is dramatically increased in
breast cancer tissues compared with normal breast tissues. Actually, overexpression of PlGF mRNA
or protein has been demonstrated in a variety of human carcinomas including breast (Parr et al.,
2005), gastric (Chen et al., 2004),lung (Zhang et al., 2005),colorectal (Wei et al., 2005), renal cell
(Takahashi et al., 1994),uterine cervical (Kodama et al., 1997), and hepatocellular carcinomas (Ho
et al., 2007).
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A significant association of higher PlGF mRNA and protein levels with regional nodal
metastasis of OSCC was shown in the present and our previous studies (Cheng et al., 2010). The
similar findings were also demonstrated in human breast cancer (Parr et al., 2005). The reasons why
PlGF increased cancer metastasis could be explained as follows. First, angiogenesis is a pivotal
factor for tumor growth and metastasis (Carmeliet et al., 2000). PlGF can promote vessel growth
and maturation directly by affecting endothelial and mural cells, as well as indirectly by recruiting
pro-angiogenic cell types. PlGF can also increase the expression of VEGFA which is a potent
angiogenic factor (Fischer et al., 2008). Furthermore, PlGF displaces VEGFA from FLT1, which
liberates VEGFA and allows it to activate FLK1 (also known as VEGFR2) and augment
VEGF-driven angiogenesis (Park et al., 1994). PlGF also activates and attracts macrophages that
release angiogenic and lymphangiogenic molecules (Selvaraj et al., 2003). In addition, PlGF can
promote tumor angiogenesis, lymphangiogenesis, and the formation of the premetastatic niche
(Fischer et al., 2008). Second, PlGF promotes the growth, survival and migration of metastatic
tumor cells. Third, PlGF enhances the expression of matrix metalloproteinase 9 (MMP9) which
facilitates the cancer cell invasion and metastasis (Fischer et al., 2008). Fourth, PlGF directly
regulates the motility of human non-small cell lung cancer cells (Chen et al., 2008) and also
stimulates in vitro motility and invasion of the human breast tumor cell lines (Taylor et al., 2007).
Moreover, an antagonistic PlGF/FLT1 peptide can inhibit the growth and metastasis of human
breast cancer xenografts (Taylor et al., 2007). Fifth, PlGF inhibits the differentiation of dendritic
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cells and in turn suppresses the antigen recognition and anti-tumor immune defense (Fischer et al.,
2008). In summary, PlGF may promote lymph node metastases through multiple mechanisms such
as an increase in tumor angiogenesis and lymphangiogenesis, an increase in tumor cell survival,
motility, migration and invasion, an elevated expression of MMP9, and an inhibition of the immune
surveillance by dendritic cells.
This study showed a significant association of PlGF mRNA overexpression in OSCCs with
larger tumor size and positive lymph node metastasis. Because higher T and N statuses always
result in more advanced clinical stages of OSCC, it is not difficult to explain why OSCCs with the
higher expression of PlGF mRNA are prone to have the more advanced clinical stages. Indeed, high
expression of PlGF mRNA or protein is significantly associated with an advanced clinical stage of
gastric (Chen et al., 2004), lung (Zhang et al., 2005),and colorectal cancers (Wei et al., 2005).
A significant correlation between the higher PlGF mRNA expression (higher -∆CT value) in
OSCCs and the poorer recurrence-free survival in OSCC patients was demonstrated in this study. In
addition, PlGF mRNA -∆CT value > 2 was identified as an independent unfavorable prognosis
factor by multivariate analyses with Cox regression model. Previous study also showed a significant
association of higher level of PlGF mRNA with a shorter survival in patients with colorectal
carcinoma (Wei et al., 2005). In addition, an increased level of PlGF protein is significantly related
to poor prognosis in patients with breast (Parr et al., 2005) or gastric carcinomas (Chen et al., 2004).
Furthermore, high PlGF protein or mRNA level is significantly associated with recurrence of breast
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cancer (Parr et al., 2005).PlGF protein or mRNA level can also predict the early recurrence after
radical resection of hepatocellular carcinoma (Ho et al., 2007).The above findings indicate that
PlGF protein or mRNA level may be an important prognostic indicator for patients with certain
types of human carcinomas including OSCC.
Our results showed a significant increase in the PlGF mRNA expression in OSCC tissues
compared to that in non-OSCC tissues. We also found that the PlGF mRNA level in OSCC samples
was significantly correlated with tumor size, N status, clinical staging, and loco-regional recurrence
of OSCCs. Moreover, OSCC patients with a higher PlGF mRNA expression had a poorer
cumulative recurrence-free survival than those with lower PlGF mRNA levels. PlGF mRNA -∆CT
value > 2 was also identified as an independent unfavorable prognosis factors for OSCCs by
multivariate analyses. These results indicate that the PlGF mRNA level may be a biomarker for
prediction of the progression of OSCCs and the prognosis of OSCC patients in Taiwan.
Part II: Increased serum placenta growth factor level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma
We recently found that the higher PlGF protein expression in OSCC tissues is significantly
associated with the more advanced tumor progression and shorter patient survival. Because PlGF is
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a soluble peptide, its serum level may indicate the angiogenic activity of the tumor and may serve as
a predictive biomarker for cancer progression, recurrence and prognosis. In this study, we measured
the pre-surgery and post-surgery serum PlGF protein levels in 72 OSCC patients and found that the
higher serum PlGF protein level could return to near normal control value 3 months after total
excision of the cancer. The prominent rise of serum PlGF protein value in pre-surgery OSCC
patients and the significant fall of serum PlGF protein value after surgical removal of tumor tissue
indicate that the OSCC tissue is the major source of PlGF proteins. Our recent study showed the
expression of PlGF in OSCC tumor cells with the mean labeling index of 51 ± 19 (Cheng et al.,
2010).Thus, at least part of the cancer cell-produced PlGF proteins may secrete into stromal tissue,
diffuse into adjacent vascular or lymphatic vessels, and drain into systemic circulation, finally
resulting in an increase in serum PlGF protein levels in pre-surgery OSCC patients. A previous
study demonstrated a persistent increase in plasma PlGF protein levels on postoperative day 1, 3,
and 7-20 after minimally invasive resection of colon cancer. The plasma PlGF protein level dropped
to the preoperative baseline level on postoperative day 21-27 (Shantha ei al., 2011). Although the
cause of the postoperative increase in plasma PlGF protein level was unclear, this study also
suggested that the cancer tissue is the major source of PlGF proteins.
In addition, the higher pre-surgery serum PlGF protein levels were significantly associated
with larger tumor size, positive lymph node metastasis, more advanced clinical stages, loco-regional
recurrence, and poorer recurrence-free survival in this study. This suggests that the serum PlGF
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protein level can also predict the progression, recurrence and prognosis of OSCC. Indeed, an
elevated serum PlGF protein level has been reported in a variety of human carcinomas including
renal cell (Matsumoto et al., 2003), pancreatic (Chang et al., 2008; Sabbaghian et al., 2010),and
colorectal carcinomas (Rahbari et al., 2011; Wei et al., 2009). Furthermore, the plasma PlGF protein
level is an independent prognostic factor for renal cell carcinoma (Matsumoto et al., 2003), and
higher preoperative serum PlGF protein levels are associated with higher risk of recurrence and
poorer prognosis of stage III colorectal cancer (Wei et al., 2009). However, Chang et al. (2008)
showed no correlation between the serum PlGF protein concentration and overall survival in
patients with pancreatic cancer. Rahbari et al. (2011) found that circulating PlGF protein levels were
correlated inversely with recurrence-free survival in patients undergoing curative resection of
colorectal liver metastases by multivariate analysis.
To the best of our knowledge, this is the first report demonstrating that serum PlGF protein
levels are significantly correlated with the progression, recurrence and prognosis of OSCC. There
are several reasons that can explain why the higher serum PlGF protein level is significantly
associated with larger tumor size and positive lymph node metastasis. First, angiogenesis is a key
factor for tumor growth and metastasis (Carmeliet et al., 2000). PlGF can stimulate vessel growth
and maturation directly by increasing the proliferation and differentiation of endothelial and mural
cells, as well as indirectly by recruiting pro-angiogenic cell types (Fischer et al., 2008). Second,
PlGF can activate and attract macrophages that release angiogenic and lymphangiogenic factors
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(Selvvaraj et al., 2003). Third, PlGF promotes the growth, survival and migration of metastatic
tumor cells (Fischer et al., 2008). Fourth, PlGF increases the expression of matrix metalloproteinase
9 which facilitates the cancer cell invasion and metastasis. Fifth, PlGF inhibits the differentiation
of dendritic cells and subsequently suppresses the antigen recognition and anti-tumor immune
defense. Therefore, the serum PlGF protein may act as a hormone-like molecule that circulates to
appropriate sites and in turn augments tumor angiogenesis and lymphangiogenesis, recruits
angiogenic and lymphangiogenic factors-producing macrophages to the tumor microenvironment,
increases tumor cell survival, migration and invasion, and inhibits anti-tumor immune activity,
finally promoting the OSCC cell growth and metastasis (Fischer et al., 2008).
PlGF expression increases significantly in early gestation, peaks at around 26-30 weeks, and
decreases as term approaches (Torry et al., 1998). The reduced PlGF mediates the genesis of
preeclampsia and cigarette smoking can reduce the risk of preeclampsia (Mehendale et al., 2007).
The serum PlGF protein levels have been reported to be significantly higher among smokers
compared to non-smokers among diabetic women and women with a history of preeclampsia
(Jeyabalan et al., 2010). Moreover, the serum PlGF protein levels are significantly higher in
smokers with chronic obstructive pulmonary disease than in control smokers without chronic
obstructive pulmonary disease and control non-smokers (Cheng et al., 2008). The results of
aforementioned studies suggest that cigarette smoking can increase the secretion of PlGF proteins
into the blood stream (Cheng et al., 2008; Jeyabalan et al., 2010; Mehendale et al., 2007). Although
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the difference was not significant, our previous study showed a slightly elevated expression of PlGF
protein in OSCC tissues of areca quid chewers and smokers (Cheng et al., 2008) and the present
study also revealed a slightly increased serum PlGF protein level in areca quid chewers and
smokers compared to non-chewers and non-smokers. Further studies are needed to clarify the exact
relation between areca quid chewing and PlGF protein expression or secretion.
Our results showed a significantly higher serum PlGF protein levels in pre-surgery OSCC
patients than in normal control subjects. Serum PlGF protein levels dropped to near the normal
control levels after surgical removal of the cancer. We also found that higher pre-surgery serum
PlGF protein levels were significantly associated with T status, N classification, clinical staging,
and loco-regional recurrence. Moreover, OSCC patients with a higher serum PlGF protein level had
a significantly poorer cumulative recurrence-free survival than those with a lower serum PlGF
protein level. We conclude that the serum PlGF protein level may be a valuable biomarker for
prediction of therapeutic effect, progression, recurrence, and prognosis of OSCC.
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