Gene Therapy for Thyroid Cancer: Current Status and Future Prospects

Christine Spitzweg; John C. Morris

Disclosures

Thyroid. 2004;14(6) 

In This Article

Corrective Gene Therapy

Most poorly differentiated thyroid tumors have lost expression of the normal p53 tumor suppressor gene through inactivating mutations. p53 is a transcription factor mediating critical cellular responses, including cell cycle arrest and apoptosis, after exposure to DNA-damaging stimuli.[2] Mutations in the p53 gene seem to be late genetic events associated with loss of differentiation and are, at least in part, responsible for the aggressive behaviour of advanced and/or dedifferentiated tumors. Restoration of wild-type (wt) p53 expression has recently been used in a variety of experimental cancer models including thyroid cancer and has been tested in human clinical trials.[3,4,5,6,7,8,9] p53 restoration has been shown to be associated with a bystander effect, which means that not only p53-transduced cells are killed, but also that surrounding nontransduced cells are killed by the transduction of their neighbors. A bystander effect is highly desirable for a therapeutic gene, because it reduces the level of transduction efficiency required for successful gene therapy, which represents one of the crucial technical hurdles for in vivo gene therapy applications. The bystander effect of p53 gene therapy results from its antiangiogenic effect, which is the result of downregulation of vascular endothelial growth factor (VEGF) and upregulation of thrombospondin, a potent inhibitor of angiogenesis.[2]

Several studies of p53 gene therapy of thyroid cancer have been reported. Expression of wild-type tumor suppressor gene p53 (wt-p53) in a p53-null thyroid carcinoma cell line (FRO) resulted in decreased cell growth in vitro and inhibition of tumorigenesis in vivo in nude mice. 40% of mice inoculated with p53-transfected FRO were tumor-free and 60% developed small hypovascular tumors indicating suppression of neovascularization.[4] In another study, retroviral p53 gene transfer into p53 mutant papillary thyroid cancer cells (NPA) resulted in a dose-dependent inhibition of tumor cell growth and enhanced chemosensitivity to adriamycin in vitro and in vivo.[5] Using a replication-deficient adenovirus expressing wild-type tumor suppressor gene p53 (wt-p53), Nagayama et al.[4] evaluated the therapeutic efficacy of p53 restoration in four human anaplastic thyroid cancer cell lines harboring p53 mutations (ARO, FRO, NPA, WRO) and normal human thyroid follicular cells in vitro and in vivo. Adenovirus-mediated p53 expression resulted in dose-dependent cell killing in thyroid cancer cell lines, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite their highest adenovirus infectivity. The mechanism of cell killing was shown to be apoptosis. In addition, wt-p53 expression sensitized some of the cell lines to the chemotherapeutic effect of doxorubicin (FRO and NPA cells) and 5-fluorouracil (FRO cells). In vivo experiments using FRO and NPA cell xenografts in nude mice showed inhibition of tumor growth following direct injection of the adenovirus expressing wt-p53. This effect was augmented by combination with doxorubicin, resulting in tumor regression.[6] To develop an adenoviral gene transfer system that replicates exclusively in wt-p53-deficient cancer cells thereby limiting the cytotoxic effect of a replication-competent adenovirus lacking E1B55K, Nagayama et al. used the geneinactivation strategy using a p53-regulated Cre/loxP system consisting of two recombinant adenoviruses. One contains an expression unit of the synthetic p53-responsive promoter and the Cre recombinase gene, and the other adenovirus contains two expression units: the first consists of the E1A gene flanked by a pair of loxP sites downstream of the constitutive CAG promoter, and the second consists of the E1B19K gene under the control of the cytomegalovirus (CMV) promoter. Coinfection of these two adenoviruses into p53 expressing cells leads to expression of Cre recombinase, which then excises the E1A gene that is flanked by a pair of loxP sites, thereby stopping virus replication. In cells without p53 expression, however, Cre recombinase is not expressed, the E1A gene not excised, and virus replication takes place thereby causing cell lysis.[7] Another, more recent study by Imanishi et al.,[8] indicated that the histone deacetylase inhibitor depsipeptide enhances apoptotic killing by p53 gene transfer in anaplastic thyroid cancer cell lines (FRO and WRO cells), suggesting that this combination treatment strategy might also be useful in the treatment of undifferentiated thyroid carcinomas.

Inhibition of oncogenic RET signaling by expression of a dominant-negative RET mutant, a new corrective gene therapeutic approach in medullary thyroid cancer, has been investigated by Drosten et al.[10,11] More than 95% of medullary thyroid carcinomas harbor dominant activating mutations in the RET proto-oncogene, which play a central role in the development of medullary thyroid cancer. The human RET proto-oncogene encodes for a transmembrane receptor (a common receptor for the glial cell derived neurotrophic factor family ligands) that consists of three functional domains: the extracellular ligand binding domain, the transmembrane segment and the intracellular domain formed by a tyrosine kinase. Mutations of the RET gene result in constitutive activation of RET tyrosine kinase with aberrant downstream signaling and initiation of tumor formation. Adenoviral vectors expressing dominant-negative RET mutants were used to investigate the effects of RET inhibition in TT cells (human medullary thyroid carcinoma cells). Because of amino acid changes in the extracellular domains of these dominant-negative RET mutants, which naturally occur associated with Hirschsprung's disease, the glycosylation process is disturbed resulting in hampered protein transport to the cell surface. In addition, the dominant-negative mutants dimerize with oncogenic RET protein in the endoplasmic reticulum (ER), thereby preventing expression of both dominant-negative and oncogenic RET protein on the cell surface. Using an adenoviral vector expressing dominant-negative RET under the control of a C-cell-specific synthetic calcitonin/calcitonin gene-related peptide promoter, a pronounced shift of endogenous oncogenic RETprotein localization from the cell surface to the ER was demonstrated, resulting in strong inhibition of cell viability caused by induction of apoptosis in vitro. Adenoviral mediated transfer of dominant-negative RET mutant in TT cell tumors in vivoin nude mice resulted in prolonged survival, while inoculation of ex vivo transduced TT cells in nude mice led to complete suppression of tumor growth in vivo.[10,11] In contrast to p53 restoration, inhibition of oncogenic RET expression by dominant-negative RET mutant delivery is not associated with a bystander effect requiring high levels of in vivo transduction efficiency, thereby limiting its therapeutic efficacy in vivo.

Recently, downregulated expression of tyrosine phosphatase η (PTPη), which encodes a receptor-type tyrosine phosphatase protein with tumor-suppressor activity, was demonstrated in human thyroid carcinomas. Adenovirus mediated transfer of rat PTPη resulted in inhibition of cell growth in four thyroid carcinoma cell lines (ARO, FB-1, NIM, TPC-1) in vitro and significant reduction of growth of anaplastic thyroid cancer cell (ARO) xenografts in nude mice. These data suggest that gene therapy based on restoration of PTPη function has potential in the treatment of human thyroid malignancies.[12]

Gadd45 family (growth arrest and DNA damage-inducible gene family) proteins have been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair, G2/M checkpoint control, and apoptosis. Chung et al.[13] demonstrated significantly lower levels of Gadd45γ RNA levels in anaplastic thyroid cancer cells compared with normal primary cultured thyrocytes. In addition, adenovirus-mediated reexpression of Gadd45γ significantly inhibited the proliferation of anaplastic thyroid carcinoma cells (ARO, FRO, NPA) resulting from apoptosis.[13]

Moreover, high mobility group I (HMGI) proteins are overexpressed in several human malignant tumors, and it has been demonstrated that inhibition of HMGI synthesis is capable of preventing thyroid cell transformation. Using an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas), Scala et al.[14] showed induction of programmed cell death in two human anaplastic thyroid carcinoma cell lines (ARO, FB-1) but not normal thyroid cells in vitro. ARO cell xenografts in nude mice revealed a drastic reduction in tumor size following intratumoral application of Ad-Yas. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may represent a useful treatment strategy for thyroid malignancies, in which HMGI(Y) gene overexpression is a general event.[14]

A common strategy for cytoreductive gene therapy is the suicide gene/prodrug strategy herpes simplex virus thymidine kinase/ganciclovir. Expression of herpes simplex virus thymidine kinase (HSV-tk) in tumor cells followed by administration of ganciclovir (GCV), which is phosphorylated by HSV-tk and competes with deoxyguanosine triphosphate in DNA polymerization, results in arrest of DNA synthesis and cell death.[15] To minimize extratumoral toxicity, thyroid-specific promoters, such as the thyroglobulin (Tg) promoter and the calcitonin promoter, have been used to target the suicide gene to thyroidal cells. Nishihara et al.[16] demonstrated a therapeutic effect of ganciclovir in thyroid carcinoma cell lines FRO and WRO after retrovirus-mediated HSV-tk gene transfer, which was associated with a significant bystander and radiosensitizing effect in vitro and in vivo in xenografted tumors in nude mice. In order to improve effectiveness and safety of HSV-tk/GCV gene therapy, Braiden et al.[17] applied the Tg promoter to target the HSV-tk gene to Tg-expressing thyroid carcinoma cells using a retrovirus. They showed an in vitro cytotoxic effect selectively in Tg expressing thyroid carcinoma cells (FRTC) in contrast to anaplastic thyroid carcinoma cells without Tg expression (FRO) with a significant growth inhibition in vivo in transduced FRTC tumors in nude mice.[17] This study demonstrates the ability of the Tg promoter to transcriptionally target therapeutic genes to thyroid carcinoma cells, albeit with lower efficacy than constitutive viral promoters such as the CMV early promoter. Nagayama et al.[18] therefore successfully applied the Cre/loxP system to enhance the therapeutic efficacy of the HSV-tk/GCV system driven by the Tg promoter.

Zhang et al.[19] performed adenovirusmediated HSV-tk gene transfer under the control of the Tg promoter, and demonstrated significant and selective suppression of cell growth in Tg-expressing cells in vitro with low in vivo toxicity after systemic administration of the adenovirus (no significant changes of serum transaminase levels and histologic liver abnormalities).

Takeda et al.[20] used the telomerase reverse transcriptase promoter to achieve tumor-specific HSV-tk/GCV gene therapy in undifferentiated thyroid carcinoma cells and demonstrated a tumor-specific therapeutic effect in vitro and in vivo.

The HSV-tk/GCV system has also been applied to therapy of medullary thyroid cancer. In rat medullary thyroid cancer cells (rMTC) the HSV-tk/GCV system was limited by a low bystander effect in vitro, which correlated well with the limited antitumor efficacy n vivo. Intratumoral application of an adenovirus carrying the HSV-tk gene under the control of the CMV promoter followed by GCV treatment resulted in destruction or stabilization of smaller tumors without a therapeutic effect in larger tumors.[21] These data confirm that a significant bystander effect is an important advantage for effective suicide gene therapy. Minemura et al.[22] performed C cell-specific adenoviral HSV-tk gene transfer by introduction of the HSV-tk cDNA into exon 4 of the calcitonin mini gene (exon 3-5) coupled to the calcitonin promoter taking advantage of C-cell-specific alternative RNA splicing. After alternative splicing, which only occurs in thyroid C cells and medullary thyroid cancer cells, exon 4 is joined to exon 3 resulting in C-cell-selective HSV-tk expression. In contrast, in neural cells, which also express the calcitonin gene, exons 1 to 3 are spliced to exons 5 and 6 to form calcitonin gene-related peptide (CGRP), therefore no HSV-tk expression will be induced using the adenovirus. Using this C-cell-specific strategy, significant suppression of cell growth was shown in vitro in human and rat medullary thyroid cancer cells, accompanied by reduced expression of HSV-tk in other cancer cell lines.[22] In a more recent study, Zhang et al.[23] evaluated the effectiveness of adenovirusmediated HSV-tk/GCV therapy driven by the Tg promoter (AdrTgtk/GCV) in a human Hurthle cancer cell line (XTC-1). A significant therapeutic effect was shown in vitro as well as in vivo in XTC-1 cell xenografts in BALB/c-SCID mice after intratumoral injection of AdrTgtk/GCV with low in vivo toxicity of AdrTgtk/GCV compared to an adenovirus carrying the noncell-specific CMV promoter (AdCMVtk/CGV).[23]

Tissue- or tumor-specific promoters, such as the Tg- and calcitonin-promoter, tend to be weaker than constitutively activated viral promoters, which is often a concern regarding transduction efficiency which has to be sufficiently high to yield a therapeutic effect. Strategies to enhance Tg promoter activity by treatment with histone deacetylase inhibitors and 8-bromo cyclic adenosine monophosphate (cAMP), by use of a tandemly repeated Tg core promoter, and to extend its applicability to poorly differentiated and anaplastic thyroid cancer with lost Tg expression by cotransfection with TTF-1 and PAX-8 have been evaluated with promising results.[24,25,26,27,28] Activity and tissue-specificity of the calcitonin promoter was enhanced by combination of a minimal human calcitonin promoter with multiple copies of tissue-specific enhancer elements, and by taking advantage of C-cell-specific RNA splicing as described above.[22,29,30,31]

Taken together, in vitro as well as in vivo experiments in several follicular cell-derived and medullary thyroid cancer cell lines have clearly demonstrated a therapeutic effect of the HSV-tk/GCV strategy, which therefore seems to be a promising therapeutic approach for future therapy of advanced thyroid cancer.

For a tumor to grow it must recruit blood supply through the process of angiogenesis, which is regulated by proangiogenic factors, such as basic fibroblast growth factor and VEGF, and antiangiogenic factors, such as angiostatin and endostatin. Endostatin is one of the most potent antiangiogenic factors and has been shown to effectively inhibit angiogenesis and tumor growth in a variety of in vivo models. In follicular thyroid carcinoma cells (FTC-133), recombinant endostatin significantly inhibited the growth of FTC-133 xenografts in nude mice. Xenografts derived from FTC-133 cells stably expressing endostatin following retrovirusmediated gene transfer revealed significantly lower tumor growth in vivo than parental FTC-133 cells. This effect was associated with reduced microvessel density in the tumors and decreased systemic levels of vascular epithelial growth factor. This study demonstrates the therapeutic efficacy of antiangiogenic strategies, such as application of recombinant endostatin protein and endostatin gene transfer in follicular cell-derived thyroid cancer.[32]

The cytopathic effect of an E1B gene-defective adenovirus (ONYX-015), which replicates only in tumor cells lacking functional p53 and causes cell death, was evaluated in human thyroid carcinoma cell lines (ARO, FRO, KAT-4). ONXY-015 induced cell death in these three anaplastic thyroid cancer cell lines, but not in a normal rat thyroid cell line. Moreover, growth of ARO xenograft tumors in nude mice was significantly reduced by local injection of ONYX-015. The ONYX-015 virus acted synergistically with the antineoplastic drugs doxorubicin and paclitaxel in inducing ARO and KAT-4 cell death.[33] Furthermore, ONYX-015 treatment enhanced radiation induced cell death in human anaplastic thyroid carcinoma cells in vitro and in vivo.[34] These data strongly suggest that ONYX015 may be a valid tool in the treatment of anaplastic thyroid cancer, in particular in combination with chemotherapy and/or radiotherapy.

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.

processing....