Downmodulation of TGF-α protein expression with antisense oligonucleotides inhibits proliferation of head and neck squamous carcinoma but not normal mucosal epithelial cells

Abstract

Interruption of an autocrine growth pathway involving TGF-alpha and EGFR may inhibit tumor growth and improve survival in head and neck cancer patients. We previously demonstrated that biopsy specimens and established cell lines from patients with squamous cell carcinoma of the head and neck (SCCHN) overexpress TGF-alpha and its receptor, epidermal growth factor receptor (EGFR) at both the mRNA and protein levels. Protein localization studies showed that TGF-alpha and EGFR are produced by the same epithelial cells in tissues from head and neck cancer patients further supporting a role for this ligand-receptor pair in an autocrine growth pathway. To confirm that TGF-alpha contributes to autocrine growth, we examined the effect of down regulation of TGF-alpha protein on SCCHN cell proliferation. Treatment of 6 SCCHN cell lines with antisense oligodeoxynucleotides targeting the translation start site of human TGF-alpha mRNA decreased TGF-alpha protein production by up to 93% and reduced cell proliferation by a mean of 76.2% compared to a 9.7% reduction with sense oligonucleotide (range P = 0.036-0.0001). TGF-alpha antisense oligonucleotide exposure also decreased TGF-alpha protein levels in normal oropharyngeal mucosal epithelial cells, however their growth rate was not affected. These findings indicate that TGF-alpha is participating in an autocrine signaling pathway in transformed, but not in normal mucosal epithelial cells, that promotes proliferation.

Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF‐κB and AP‐1 activation and IL‐8 and VEGF expression in human head and neck squamous cell carcinoma lines

“…9 It will be important to determine if these apparent differences in NF-Bregulated expression of IL-8 and VEGF in different tumor lines are due to direct or indirect effects of activation of NF-B on the VEGF promoter, since the VEGF promoter lacks an apparent NF-B DNA binding site. 56 The EGFR, MEK-AP-1 and PI3K-NF-B have been individually implicated in cellular proliferation and survival, 19,22,30,38,42 and EGFR, PI3K and MEK antagonists partially inhibited growth or survival of HNSCC (Fig. 7).…”

“…7,19,22,30,38,42 To determine the effects of EGFR, MEK and PI3K antagonists on cell proliferation and/or survival, we cultured HNSCC lines with PD153035, U0126 and LY294002, and determined the effect on cell density by MTT assay (Fig. 7).…”

TGF-α antisense gene therapy inhibits head and neck squamous cell carcinoma growth in vivo

“…We and others have reported inhibition of cancer cell line growth following treatment with TGF-␣ antisense oligonucleotides or an expression vector in vitro. 34,42 In our earlier study, TGF-␣ antisense oligonucleotides down-modulated TGF-␣ protein expression in both HNSCC and normal mucosal epithelial cells. However, the normal cells were not growth inhibited by TGF-␣ antisense oligonucleotide treatment suggesting that the proliferative effects of TGF-␣ are specific for transformed cells.…”

“…34 To determine the consequences of targeting TGF-␣ in vivo, we cloned a TGF-␣ antisense sequence designed against the translation initiation site of the human TGF-␣ gene into an expression vector based on the U6 small nuclear RNA (snRNA) promoter. …”

“…However, the normal cells were not growth inhibited by TGF-␣ antisense oligonucleotide treatment suggesting that the proliferative effects of TGF-␣ are specific for transformed cells. 34 Several groups have reported the consequences of TGF-␣ targeting in vivo. Implanted LE2 mouse cells transfected with a TGF-␣ antisense expression vector showed slower growth compared with that of parental cells transfected with control vector alone.…”