TGF-Α Antisense Gene Therapy Inhibits Head and Neck Squamous Cell

Gene Therapy (2000) 7, 1906–1914 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt ACQUIRED DISEASES RESEARCH ARTICLE TGF-␣ antisense gene therapy inhibits head and neck squamous cell carcinoma growth in vivo S Endo1, Q Zeng1, NA Burke2,YHe3, MF Melhem4, SF Watkins2, MN Lango1, SD Drenning1, L Huang3 and J Rubin Grandis1,2 Departments of 1Otolaryngology, 2Cell Biology and Physiology, 3Pharmacology, 4Pathology, University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Unlike normal mucosal squamous epithelial cells, head and remained localized to the nucleus for up to 3 days. Direct neck squamous cell carcinomas (HNSCCs) overexpress inoculation of the TGF-␣ antisense (but not the correspond- TGF-␣ mRNA and protein which is required to sustain the ing sense) construct into established HNSCC tumors proliferation of HNSCC cells in vitro. To determine whether resulted in inhibition of tumor growth. Sustained antitumor TGF-␣ expression contributes to tumor growth in vivo, cat- effects were observed for up to 1 year after the treatments ionic liposome-mediated gene transfer was used to deliver were discontinued. Down-modulation of TGF-␣ was an antisense expression construct targeting the human TGF- accompanied by increased apoptosis in vivo. These experi- ␣ gene into human head and neck tumor cells, grown as ments indicate that interference with the TGF-␣/EGFR subcutaneous xenografts in nude mice. The TGF-␣ anti- autocrine signaling pathway may be an effective therapeutic sense gene was immediately detected in the cytoplasm of strategy for cancers which overexpress this ligand/receptor the tumor cells, translocated to the nucleus by 12 h and pair. Gene Therapy (2000) 7, 1906–1914. Keywords: transforming growth factor alpha; head and neck cancer Introduction by the same tumor cell provides indirect evidence of an autocrine regulatory pathway. Reports suggest that the ␣ Transforming growth factor alpha (TGF- ) is a polypep- maintenance of an autocrine loop in HNSCC is depen- tide growth factor that binds exclusively to the epidermal dent on both elevated levels of EGFR and the presence 1,2 growth factor receptor (EGFR). It is synthesized as a of TGF-␣.30,31 ␣ larger, membrane-bound glycoprotein (proTGF- ), which TGF-␣ overexpression has been implicated as a poor is cleaved to release a soluble 50 amino acid polypeptide prognostic factor in cancers of the kidney32 and eso- ␣ ␣ (TGF- ). TGF- plays an important role in regulating phagus.16 The biologic importance of this growth factor growth and development of a variety of cells including in head and neck cancer progression is supported by our 3,4 5 6 7 skin, mammary epithelium, GI tract mucosa, brain, findings demonstrating that survival of HNSCC patients 8 gynecologic and reproductive organs, kidney, and correlates significantly with TGF-␣ protein expression 9 bladder. levels in the primary tumor, independent of other clinical ␣ Overexpression of TGF- alone, or in combination with and pathological parameters including the presence of EGFR, leads to colony formation in soft agar and tumor regional metastases (N-stage).33 The observation that ␣ formation in nude mice, thus linking TGF- to transform- elevated levels of TGF-␣ are associated with the loss of 10 ␣ ation. TGF- transgenic mice display evidence of epi- growth control in many cancers has led to the develop- thelial hyperplasia, pancreatic metaplasia and carcinoma ment of strategies aimed at down-modulating this 11 ␣ of the breast. Co-expression of TGF- and EGFR has growth factor in an attempt to decrease tumor cell also been implicated in the pathophysiology of human proliferation. cancer. Overexpression of TGF-␣ alone, or in combination with EGFR, has been reported in primary malignancies and cell lines established from a wide variety of tumors, Results including glioblastoma,12,13 cancers of the breast,14,15 eso- 16 17,18 19 20,21 phagus, pancreas, liver, colorectal region, We previously reported that TGF-␣ antisense oligonucle- 20,22 9,23 24 25 26 lung, kidney, bladder, skin, ovary, vulva, cer- otides inhibited the growth of HNSCC but not normal 8 27–29 vix, endometrium, as well as the head and neck. mucosal cells in vitro, suggesting that the growth stimul- Increased production of a growth factor and its receptor ating effects of TGF-␣ are specific for transformed squamous epithelial cells.34 To determine the consequences of targeting TGF-␣ in vivo, we cloned a TGF-␣ antisense Correspondence: J Rubin Grandis, Dept of Otolaryngology, University of sequence designed against the translation initiation site Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA of the human TGF-␣ gene into an expression vector based Received 31 December 1999; accepted 24 July 2000 on the U6 small nuclear RNA (snRNA) promoter. TGF-␣ antisense gene therapy for head and neck cancer S Endo et al 1907 Figure 1 Schematic representation of the TGF-␣ antisense (or sense) oligonucleotide sequences contained in the U6 expression cassette. Transduction of the TGF-␣ antisense gene into HNSCC Similar gene localization studies were performed in tumor cells in vitro HNSCC tumors in vivo. Tumors were injected and har- Experiments were performed to elucidate the conse- vested 1 week or 2 weeks following a single injection. quences of TGF-␣ down-modulation in HNSCC cells. To The antisense gene was detected in proportion of the determine the intracellular localization of the TGF-␣ antitumor cells 1 week after treatment. The labeling charac- sense gene in head and neck cancer cells, fluorecein- teristics in vivo were similar at both 1 week and 2 week labeled DNA (Figure 1) was used to tranfect the HNSCC time-points (Figure 3). cells in vitro followed by examination of the tumor cells using fluorescence microscopy at different time-points TGF-␣ antisense gene therapy inhibits tumor growth (Figure 2). By 12 h, labeling was detected within the To determine whether or not the injected gene could be nucleus of sectioned cells. Furthermore, there was no detected in the tumors, DNA was harvested from the quantitative difference in the amount of labeled DNA in treated xenografts followed by PCR using specific pri- the nucleus for up to 72 h (Figure 2b–e), demonstrating mers designed to detect the recombinant TGF-␣ sense or persistence of the antisense DNA in the cells. No label antisense gene. The recombinant DNA was detected in was detectable in cells which received fluorecein-labeled all tumors in contrast to tumors treated with liposomes liposomes alone or unlabeled DNA (data not shown). alone (Figure 4). To examine the consequences of the These results demonstrate that transduction of TGF-␣ transfected TGF-␣ antisense gene on tumor growth in antisense DNA complexed with DC-chol liposomes into vivo, established HNSCC xenografts were repeatedly head and neck tumor cells, results in nuclear localization inoculated with 50 ␮g TGF-␣ antisense DNA plus 50 of the transduced gene in tumor cells for at least 3 days nmol DC-chol liposomes. Previous studies from our lab- following transfection. oratory have demonstrated the necessity of a carrier mol- Figure 2 Intracellular localization of the TGF-␣ antisense gene in vitro. HNSCC cells transfected with FITC-tagged TGF-␣ antisense (green) and counterstained with Hoechst nuclear stain (red) were imaged by fluorescence microscopy. Nuclear localization is indicated by yellow. Five hours after transfection (a) no nuclear localization of the DNA was observed. However, at the 12 h time-point (b) a small amount of anti-sense DNA is localized to the nucleus (white arrow). By 24 h (c), nuclear localization is clearly visible and persists 48 (d) and 72 (e) h after transfection. Gene Therapy TGF-␣ antisense gene therapy for head and neck cancer S Endo et al 1908 Figure 3 Localization of antisense gene in vivo. Panel a shows a typical H&E stained image of the tumors, panel b shows the fluorescence microscope image captured before H&E staining at 1 week. Arrows in panels a and b indicate the same cells. Panel c is a representative sample showing intracellular gene 2 weeks after injection. Bar, 25 microns. Figure 4 Detection of sense or anti-sense TGF-␣/U6 gene(s) in representative HNSCC xenografts. Tumor-bearing mice were treated with intratumoral injections of TGF-␣ antisense (AS) or sense (S) DNA plus liposomes or with liposomes alone (control). PCR detection demonstrates chimeric DNA in the treated tumors as demonstrated on an ethidium bromide-stained 1% agarose gel. ecule such as DC-chol to achieve an antitumor effect.35 Dose-response experiments using a similar approach to target EGFR showed equal antitumor efficacy using 25 ␮gor50␮g of DNA per injection with less antitumor Figure 5 In vivo growth inhibition of HNSCC xenografts. The growth effects seen at lower doses (2.5 ␮g, 0.25 ␮g, 0.025 ␮g).35 inhibitory effects of the TGF-␣ antisense construct in a representative In contrast to tumors treated with TGF-␣ sense DNA plus experiment is demonstrated. Groups of mice received intratumoral treatments (three times a week) with TGF-␣ antisense DNA plus liposomes liposomes or tumors treated with liposomes alone, ᭺ ␣ ȣ ␣ ( ), TGF- sense DNA plus liposomens ( ), or liposomes alone ( ) 14– HNSCC xenografts inoculated with TGF- antisense 21 days following tumor implantation. All cases received nine treatments. DNA plus liposomes were growth inhibited (Figure 5). Each point represents the mean value for eight to 10 tumors from an To determine the persistence of the antitumor effects, a individual experiment that was replicated three times. Fractional tumor subset of mice treated with the TGF-␣ antisense construct volume (tumor volume as a proportion of pretreatment volume) is plotted plus liposomes were maintained and followed for up to and the standard error of tumor volumes for all points was less than 10% 1 year after the treatments were discontinued. In these of the mean.

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