The close association between high HA level and malignancy has been reported in many different types of cancer including breast, ovarian, and bladder cancers [3
]. However, there is only limited information regarding the contribution of HA in the development of NSCLC. Existing information suggests that HA might have a contributing role in this process [2
The synthesis of HA is a tightly regulated process and is modulated by cytokines and growth factors in a number of different cancer cells [8
]. Cytokines and growth factors are found in the lung tumor microenvironment and are derived from tumor cells as well as stromal and inflammatory cells [42
]. In this paper, we investigated the effect of EGF, IL-1β
1 in modulating HA synthesis in A549 cells, a lung adenocarcinoma cell line. These factors were chosen as they have been reported to play a role in the progression of lung cancer as elevated expression of EGF, IL-1β
, and TGF-β
1 have been reported in patients with lung cancer [48
]. These factors either separately or in combination was able to stimulate HAS2 transcript more than HAS3 transcript () suggesting that HAS2 mRNA is more responsive to external stimuli which has also been observed in human mesothelial cells [51
]. However, induction of HAS isoform expressions resulted in an increase in HA production that mimic the up-regulation of HAS3 isoform, with TGF/IL combination giving the highest stimulation (), suggesting a dominant role for HAS3 isoform.
Increased in HA production resulted in a HA-dependent pericellular matrix in TGF/IL cultures () that could participate in biological processes pertaining to migration and proliferation as these HA-rich matrices have been reported in migrating and proliferating cells [52
]. In addition, TGF/IL treatment also resulted in morphological and biological changes whereby there is the combined loss of staining for epithelial marker, E-cadherin, and a gain in mesenchymal marker, vimentin () indicating a shift to a mesenchymal-like phenotype. This finding suggests that HA synthesis can be modulated by various cytokines and growth factors in NSCLC. Increase in HA level might play a role in the EMT-like transition in NSCLC as HA has been shown to be involved in EMT in normal cardiac development [38
To define the role of HA in EMT-like process in NSCLC, we overexpressed HAS3 gene in H358 cells as these cells exhibit a more epithelial phenotype as compared to A549 [33
]. Overexpression of HAS3 gene in H358 resulted in a change is morphology from a more compact and cobblestone-like appearance to cells that are elongated in shape with a concomitant appearance of vimentin expression and a reduced staining for E-cadherin, indicating a gain in mesenchymal-like phenotype (). Our finding is in agreement with a report that showed HA can induced EMT-like transition and acquisition of transformed characteristics in normal epithelial cells [39
]. Moreover, it has been reported that the HA-CD44 axis plays an important role in morphogenesis in mammalian organogenesis. In this context, overexpression of E-cadherin, in cells with low levels of E-cadherin expression, negatively modulates HA interaction with its receptor, CD44, that leads to the inhibition of CD44-mediated cell invasion and branching morphogenesis [40
]. Our study indicates that increase in HA production can modulate cell surface expression of E-cadherin and promote a shift to a mesenchymal-like phenotype in NSCLC. Therefore, this result suggests that there is a cross-talk between E-cadherin and HA regulation. Although the mechanism by which HA mediates this effect in NSCLC remains to be studied it may be possible that anomalous overexpression of HA destabilizes the balance in normal epithelial cells with high levels of E-cadherin expression and induces a shift to an EMT differentiation process.
One of the characteristic of cells that have undergone EMT is the acquisition of an invasive phenotype [27
]. We observed that overexpression of HAS3 gene in H358 resulted in the cells were more invasive compared to vector-only control cells (). MMP2 and MMP9 have been implicated to play a role in cell invasion and metastasis as they are involved in the turnover of extracellular matrix components in the basement membrane [44
]. In our system, both MMP9 and MMP2 transcripts were elevated with the MMP9 mRNA expression being more abundant () and this was reflected by an increased in their activities as assessed by the gelatin zymography (). Our data supports that increased HA production is able to stimulate the production of MMP2 and MMP9 leading to a more invasive phenotype in NSCLC cell line. This is in line with a recent report that demonstrated that normal epithelial cells that have undergone EMT following overexpression of HAS2 gene also induced elevated expressions of MMP9 and MMP2 [39
]. Further, treatment with exogenous HA was also able to stimulate MMP2 secretion in small cell lung cancer [25
EGFR is overexpressed in human cancer including NSCLC and is correlated with poor prognosis [46
]. EGFR-TKIs have been used in the treatment of NSCLC that produced a response rate of 9 to 27% [53
]. To improve the treatment outcome, extensive investigations have been carried out to identify the underlining causes for the low response rate [56
]. One such finding indicated that EMT is a determinant of sensitivity of NSCLC to EGFR inhibition whereby tumor cells exhibiting mesenchymal-like phenotypes were more resistant to EGFR-TKI [33
] which could be attributed to kinase switching to alternate autocrine signaling thereby attenuate the dependence of EGFR signaling [58
]. Further, a study showed that restoring E-cadherin expression in EGFR-TKI resistance cells increased sensitivity to EGFR inhibition [37
] that implies EMT has a contributing role in EGFR-TKI resistance. Additional data to support this hypothesis was from a study that reported that patients with epithelial-like NSCLC that showed strong E-cadherin staining exhibited a significant longer time to progression [34
]. Our finding showed that H358 that have been induced to undergo EMT-like transition by overexpression of HAS3 were more resistant to EGFR-TKI treatment, as compared to vector-only control. There was no detectable cell death at 10μ
M EGFR-TKI in the HAS3 transfectants while control cells growth rate was reduced by 40% (). Similarly, addition of exogenous HA to A549 cells showed more resistance to EGFR-TKI and this resistance was abolished with coincubation with Streptomyces
hyaluronidase indicating the role of HA in promoting resistance to EGFR-TKI (). Studies have also documented that interaction between HA and its receptor, CD44 induces chemoresistance in NSCLC [23
] as well as in myeloma cells [22
] and promote survival. Thus, this implies that HA can promote cell survival by inducing drug resistance to both EGFR-TKI and chemotherapeutic drugs.
In conclusion, our result suggests that HA is involved in EMT in NSCLC. Overexpressing HAS3 in H358 cells results in loss of immunoreactivity for E-cadherin and gain of vimentin expression. These cells also showed enhanced MMP2 and MMP9 activities and were more invasive. Finally, these cells were also more resistance to treatment with EGFR-TKI. In addition, we also demonstrated that various cytokines and growth factors can stimulate HA production in a NSCLC cell line that could be a contributing factor in the EMT-like transition observed in the cytokines-treated cultures. Taken together, these results support that HA can regulate epithelial to mesenchymal-like transition and therefore contribute to tumorigenesis. Thus, the control of HA level may be a potential target for therapeutic intervention for managing this disease.