Aberrant glycosylation frequently occurs in cancers and plays a critical role in
cancer progression, angiogenesis and metastasis. Common feature of tumors is the
overexpression of truncated O-glycans, such as the disaccharide Thomsen–Friedenreich
antigen (T antigen), the monosaccharide GalNAc (also known as Tn) and their
sialylated forms (ST and STn). Specifically, STn expression modulates malignant
phenotypes in gastric and breast cancer cells, such as increased migration and
invasion.[6,7,34] Altered expression of glycans can be attributed to abnormal
expression of glycosyltransferases. In previous studies, GALNT2 altered the expression
of Tn antigen on EGFR in oral squamous cell carcinoma and hepatocellular carcinoma
and Tn antigen on MET in gastric cancer. [14,15] GALNT10 modified O-glycosylation
of EGFR and subsequent phosphorylation of Akt in HBV-associated HCC.[35]
Besides, GALNT2 could modify O-glycosylation and activation of IGF-1R, and then
affect the malignant phenotypes of neuroblastoma cells.[36] However, no studies
investigate about the role of GALNT2 in modifying O-glycosylation of EGFR in gastric
cancer. Here, we demonstrated that knockdown of GALNT2 decreased the Tn antigen
on EGFR in gastric cancer. Since alterations in glycosylation can significantly impact
overall glycoprotein charge and conformation and therefore readily alter its biological
activity. For example, aberrant glycosylation of receptor tyrosine kinases (RTKs) could
modulate their activities and signalings. [7] Receptor tyrosine kinase (RTK) -targeted
therapeutic agents are constantly being developed and have been shown to be effective in
various clinical trials. Trastuzumab, an inhibitor of ERBB2, has been approved for the
treatment of gastric cancer.[37] So investigating the roles of other RTKs in gastric cancer
will provide potential targets for the future treatments. In our previous study, we revealed
that GALNT2 knockdown increased EGFR phosphorylation. Constitutive activation of
the EGFR, which can be resulted from ligand depend or ligand independent pathway is
common in cancers [38] Here, we showed that knockdown of GALNT2 enhanced EGFR
phosphorylation in the presence of EGF. On the other hand, without EGF, GALNT2
didn’t affect EGFR phosphorylation. This may indicate EGFR activation modulated by
GALNT2 may dependent on the presence of ligand. Previous researches showed
glycosylation of EGFR might affect their bind affinity to EGF [39,40]. The mechanism
of how GALNT2 affects EGFR phosphorylation needs further study.
Recent studies suggested that GALNT2 regulated the malignant phenotypes by
modifying EGFR glycosylation and phosphorylation in oral squamous carcinoma and
hepatocellular carcinoma.[14,15] This study demonstrated the migration and invasion
abilities of AGS cells enhanced by GALNT2 would be suppressed by inhibiting EGFR
phosphorylation. Subsequently, inhibition of EGFR phosphorylation decreased
expression of phospho-Akt. Previous studies revealed that EGFR promoted gastric
cancer migration and invasion via Akt-activation.[27,28] In this study, cell viability,
migration and invasion abilities of AGS cells were significantly suppressed by inhibiting
Akt phosphorylation. These findings reveal that EGFR-Akt activation plays an important
role in gastric cancer progression.
In present study, cell viability during day1-5 was not affected by GALNT2 and
EGFR activation but Akt activation. In contrast to our previous study, knockdown of
GALNT2 enhanced cell viability.[33] This may be because this difference was
significant at day 6 in previous study, but we only assessed cell viability for 5 days.
Current studies about the effect of gefitinib on cell growth inhibition are controversial.
Gefitinib inhibited proliferation in prostate cancer and lung cancer.[41] However, it was
ineffective against most EGFR wild-type non-small cell lung cancer, but combined
EGFR siRNA or Akt inhibitior showed synergistic growth inhibition. [42,43]
However, there is no research about correlation of GALNT2 and phospho-EGFR in
clinical samples. This is the first study to investigate this relationship, but result showed
p-EGFR positively correlated with GALNT2, which was opposite to in vitro experiments.
The possible reasons may be as followed: first, the limitation of IHC analysis of
detection of two proteins simultaneously, so two sections were used to detect expression
of GALNT2 and phospho-EGFR respectively, which might result in scoring at different
cells. Although, we minimize this limitation by sectioning serially, but the final slide for
evaluated might not from the serial section. Immunofluorescence may serve as method to
solving this problem by double labeling of different proteins in the same slide.[44,45]
Second, downregulation of GALNT2 enhances the malignancy in gastric cancer may
attribute to multiple pathways, not all the patients undergo increasing EGFR
phosphorylation, so it needs further investigation of subgroup analysis, and it might
demonstrate clinical significance.
EGFR becomes activated upon phosphorylated, so increasing numbers of studies on
correlation of p-EGFR with clinical outcomes. Patients with p-EGFR positive exhibited
poorer overall survival.[30,31], but Nieto et al. showed p-EGFR didn’t serve as a
prognostic marker for overall survival.[29] The prognostic role of p-EGFR in gastric
cancer remains unknown. In present study revealed there were no difference in
clinico-pathalogical parameters, such as tumor size, cell differentiation, TNM stage and
progression-free survival between p-EGFR positive and negative. The plausible
explanations for controversial findings are as followed. First, scoring criteria and
threshold for positive and negative applied in each papers are not equal. Second, there
are many phosphorylation sites of EGFR, such as pY845. pY1068 and pY1173 etc., and
initiating distinct downstream signaling pathways. Recent studies detected not the same
site, and it might result in different outcomes. Third, gefitinib inhibit overall
phosphorylation of EGFR, not site specific, so other tyrosine sites may play a more
critical role in enhancing malignancy in gastric cancer. In addition, distinct molecular
drivers and tumor biology, and thus different treatment targets and predictive biomarkers
are implicated in each subtype of gastric cancer, such as intestinal versus diffuse type.
For example, EGFR, HER2 and MET overexpression are more prevalent in intestinal
type, but loss of E-cadherin and FGFR overexpression are more common in diffuse
type.[46,47] In this study, only intestinal type samples were collected for analyzing.
Thus, we will collect diffuse type samples to investigate if p-EGFR serves as a
prognostic marker in this subtype.
In conclusion, our findings of GALNT2 regulating malignancy by modifying
O-glycosylation and phosphorylation of EGFR brings new insights into new therapeutic
strategies. However, the prognostic role of pEGFR needs further investigation.