Inhibition of tumor growth and invasion by YC-1 in nasopharyngeal carcinoma (NPC) (2007)

B197 Objective: Nasopharyngeal carcinoma (NPC) is highly prevalent in Southeast Asia. The majority of NPC deaths are due to metastasis. Hypoxia is known to contribute to cancer progression and metastasis in various human malignancies, including NPC. Hypoxia-inducible factor 1-alpha (HIF-1α) plays a major role in promoting cancer growth, metastasis and angiogenesis. We have previously shown that HIF-1α and several related hypoxia markers (CA IX and VEGF) were over-expressed in about 60% of NPC cases and over-expression of HIF-1, CA IX and VEGF are associated with poor survival in NPC patients (Hui et al., Clin. Cancer Res., 2002). In this study, we investigated the effects of YC-1, a compound known to inhibit HIF-1α, on NPC cell growth and invasion. We hypothesize that HIF-1α targeting would be elicit antitumor activity in NPC models. Methods: Three human NPC cell lines, HONE-1, CNE-2 (derived from poorly differentiated NPC) and HK1 (from well-differentiatedNPC) were used. Hypoxia treatment: cell incubation in a hypoxia incubator (0.1% O2, 5% CO2, 94.9% N2). The effects of YC-1 on NPC cell growth was assessed by MTT assay, while its effects on NPC invasion was determined by wound healing assay as well as the Matrigel Invasion assay. Western blotting was employed for the study of the effects of YC-1 on NPC signaling. Results: YC-1 effectively induced a dose-dependent growth inhibition of a NPC cell line, HONE-1 (IC50= 535.6 ± 64.6 nM, 48 hrs). YC-1 treatment at 10 µM resulted in 71.6 ± 0.4 % and 47.0 ± 1.8 % growth inhibition in HONE-1 and CNE-2, respectively. However, no detectable growth inhibition was observed in HK1 cell when treated with equal concentration of YC-1. Using a representative NPC cell line, HONE-1, we also demonstrated that YC-1 was able to effectively inhibit the hypoxia-induced HIF-1α up-regulation in a dose-dependent manner. However, YC-1 did not preferentially enhance NPC growth inhibition under hypoxia in all three NPC cell lines. We also found that YC-1 inhibited NPC motility and invasive migration in vitro. YC-1, at both 350 nM and 700 nM, inhibited the spontaneous motility of HONE-1 cells in the absence of serum. Serum-induced invasive migration of HONE-1 through the Matrigel Invasion chamber was also significantly inhibited by YC-1 at 250 nM (~60% inhibition when compared to the vehicle control p=0.0007). It is known that epidermal growth factor receptor (EGFR) signaling plays an important role in NPC growth and invasion. However, the relationship between HIF-1α and EGFR in NPC is unclear. In an NPC cell line, HONE-1, hypoxia not only induced HIF-1α up-regulation, but also EGFR up-regulation (at 24 hrs and 48 hrs). Stable clones expressing EGFR shRNA greatly attenuated the hypoxia-induced HIF-1α up-regulation (at 6 hrs of hypoxia treatment). Our results indicated that HIF-1α and EGFR expression are coupled in NPC by unknown mechanism. Combined targeting of EGFR (by an EGFR-targeting monoclonal antibody, C225, 100 nM) and YC-1 (500 nM) resulted in additive growth inhibition of HONE-1 cells in both normoxic and hypoxic conditions (at 48 hrs). Conclusions: In summary, our data demonstrated that YC-1 targeting is effective in inhibiting NPC growth and invasion. Our data also suggest that HIF-1α and EGFR signaling seem to be coupled in NPC cells. Combination of YC-1 and EGFR monoclonal antibody resulted in enhanced antitumor effects on NPC cells.  Supported by RGC of HKSAR (project no: CUHK 4442/06M)