Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
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Animals↗
Biomedical subjects
D Hanahan
Publications and source records attributed to D Hanahan.
Chronic estrogen-induced cervical and vaginal squamous carcinogenesis in human papillomavirus type 16 transgenic mice.
High-risk human papillomaviruses (HPVs), including type 16, have been identified as factors in cervical carcinogenesis. However, the presence and expression of the virus per se appear to be insufficient for carcinogenesis. Rather, cofactors most likely are necessary in addition to viral gene expression to initiate neoplasia. One candidate cofactor is prolonged exposure to sex hormones. To examine the possible effects of estrogen on HPV-associated neoplasia, we treated transgenic mice expressing the oncogenes of HPV16 under control of the human keratin-14 promoter (K14-HPV16 transgenic mice) and nontransgenic control mice with slow release pellets of 17beta-estradiol. Squamous carcinomas developed in a multistage pathway exclusively in the vagina and cervix of K14-HPV16 transgenic mice. Estrogen-induced carcinogenesis was accompanied by an incremental increase in the incidence and distribution of proliferating cells solely within the cervical and vaginal squamous epithelium of K14-HPV16 mice. Expression of the HPV transgenes in untreated transgenic mice was detectable only during estrus; estrogen treatment resulted in transgene expression that was persistent but not further upregulated, remaining at low levels at all stages of carcinogenesis. The data demonstrate a novel mechanism of synergistic cooperation between chronic estrogen exposure and the oncogenes of HPV16 that coordinates squamous carcinogenesis in the female reproductive tract of K14-HPV16 transgenic mice.
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Antiangiogenic therapy of transgenic mice impairs de novo tumor growth.
Angiogenesis is activated during multistage tumorigenesis prior to the emergence of solid tumors. Using a transgenic mouse model, we have tested the proposition that treatment with angiogenesis inhibitors can inhibit the progression of tumorigenesis after the switch to the angiogenic phenotype. In this model, islet cell carcinomas develop from multifocal, hyperproliferative nodules that show the histological hallmarks of human carcinoma in situ. Mice were treated with a combination of the angiogenesis inhibitor AGM-1470 (TNP-470), the antibiotic minocycline, and interferon alpha/beta. The treatment regimen markedly attenuated tumor growth but did not prevent tumor formation; tumor volume was reduced to 11% and capillary density to 40% of controls. The proliferation index of tumor cells in treated and control mice was similar, whereas the apoptotic index was doubled in treated tumors. This study shows that de novo tumor progression can be restricted solely by antiangiogenic therapy. The results suggest that angiogenesis inhibitors represent a valid component of anticancer strategies aimed at progression from discrete stages of tumorigenesis and demonstrate that transgenic mouse models can be used to evaluate efficacy of candidate antiangiogenic agents.
Adenoma, Islet Cell↗
Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice.
Immune surveillance depends on lymphocyte access to tissue. Lymphocytes emigrate from blood when adhesion receptors such as L-selectin and the alpha 4 beta 7 integrin on these cells bind to ligands expressed on venular endothelium. Among transgenic mouse lines expressing an oncoprotein (Tag) in islet beta cells, some recognize Tag as nonself. In these mice, Tag expression elicits both beta cell hyperplasia with subsequent progression to tumors and lymphocytic infiltration. Endothelial ligands for L-selectin and alpha 4 beta 7 were upregulated in infiltrated islets in these transgenic mice. These ligands were not expressed in tumors, which were devoid of lymphocytic infiltration. In contrast, the adhesion molecules PECAM-1, ICAM-1, and VCAM-1 were expressed on endothelium in both noninfiltrated tumors and infiltrated islets. Thus, upregulation of expression of endothelial ligands for L-selectin and alpha 4 beta 7 may contribute to autoimmune infiltration. Repression of expression of these same ligands may be involved in the failure of tumor immunity.
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Transgenic mouse models of tumour angiogenesis: the angiogenic switch, its molecular controls, and prospects for preclinical therapeutic models.
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Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis.
Telomeres consist of short repeated sequences that are synthesized by telomerase, a ribonucleo-protein DNA polymerase. Telomerase activity is present in many tumours and not detected in many normal tissues. Telomere shortening in human and mouse tissues and primary cell cultures may be due to the absence of telomerase activity. To determine when telomerase is activated during tumour development and progression, we examined telomerase activity and expression of the recently cloned mouse telomerase RNA component (mTR) in two different transgenic mouse models of multi-stage tumorigenesis. These mouse models allow examination of many independent tumours from genetically identical individuals. These mice reproducibly develop pancreatic islet cell carcinoma and squamous cell carcinoma of the skin. In both carcinoma types, we detected telomerase activity only in late-stage tumours; in contrast, we found mTR levels were upregulated in the early preneoplastic stages, and further increased during progression. Surprisingly, mTR levels did not parallel the amount of telomerase activity detected and a subset of tumours lacked telomerase activity and yet expressed telomerase RNA. Regulation of telomerase activity may therefore be separable from expression of its RNA component. These results clearly demonstrate telomerase is activated in late stages of tumour progression, and show for the first time that the initial up regulation of telomerase RNA is an early event, concurrent with the hyperproliferation elicited by viral oncogenes.
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Cellular transformation and malignancy induced by ras require c-jun.
ras is an important oncogene in experimental animals and humans. In addition, activated ras proteins are potent inducers of the transcription factor AP-1, which is composed of heterodimeric complexes of Fos and Jun proteins. Together with the fact that deregulated expression of some AP-1 proteins can cause neoplastic transformation, this finding suggests that AP-1 may function as a critical ras effector. We have tested this hypothesis directly by analyzing the response to activated ras in cells that harbor a null mutation in the c-jun gene. The transcriptional response of AP-1-responsive genes to activated ras is severely impaired in c-jun null fibroblasts. Compared with wild-type cells, the c-jun null cells lack many characteristics of ras transformation, including loss of contact inhibition, anchorage independence, and tumorigenicity in nude mice; these properties are restored by forced expression of c-jun. Rare tumorigenic variants of ras-expressing c-jun null fibroblasts do arise. Analysis of these variants reveals a consistent restoration of AP-1 activity. The results provide genetic evidence that c-jun is a crucial effector for transformation by activated ras proteins.
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Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors.
Pancreatic organogenesis has been a classic example of epitheliomesenchymal interactions. The nature of this interaction, and the way in which endocrine, acinar and ductal cell lineages are generated from the embryonic foregut has not been determined. It has generally been thought that mesenchyme is necessary for all aspects of pancreatic development. In addition islets have been thought to derive, at least in part, from ducts. We microdissected 11-day embryonic mouse pancreas and developed several culture systems for assays of differentiation: (i) on transparent filters; (ii) suspended in a collagen I gel; (iii) suspended in a basement membrane rich gel; (iv) under the renal capsule of an adult mouse. Epithelia were grown either with or without mesenchyme, and then assayed histologically and immunohistochemically. Epithelium with its mesenchyme (growth systems i-iv) always grew into fully differentiated pancreas (acinar, endocrine, adn ductal elements). In the basement membrane-rich gel, epithelium without mesenchyme formed ductal structures. Under the renal capsule of the adult mouse the epithelium without mesenchyme exclusively formed clusters of mature islets. These latter results represent the first demonstration of pure islets grown from early pancreatic precursor cells. In addition, these islets seemed not to have originated from ducts. We propose that the default path for growth of embryonic pancreatic epithelium is to form islets. In the presence of basement membrane constituents, however, the pancreatic analage epithelium appears to be programmed to form ducts. Mesenchyme seems not to be required for all aspects of pancreatic development, but rather only for the formation of acinar structures. In addition, the islets seem to form from early embryonic epithelium (which only express non-acinar genes). This formation occurs without any specific embryonic signals, and without any clear duct or acinus formation.
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Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.
Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.
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Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis.
c-Myc and Mad each form heterodimers with Max that bind the same E-box related DNA sequences. Whereas Myc:Max complexes activate transcription and promote cell proliferation and transformation, Mad:Max complexes repress transcription and block c-Myc-mediated cell transformation. Here we examine these antagonistic transcription factors during epithelial differentiation and neoplastic progression. During differentiation of primary human keratinocytes, Mad is rapidly induced and c-Myc is downregulated, resulting in a switch from c-Myc:Max to Mad:Max heterodimers. In normal epidermis and colonic mucosa c-myc expression is restricted to proliferating cell layers, while mad expression is restricted to differentiating cell layers. Using HPV18 transformed keratinocytes that vary in their ability to differentiate in organotypic cultures, we find that Mad induction occurs only in those cells that retain a differentiation response. In the epidermis of transgenic mice in which expression of the HPV16 E6 and E7 oncogenes are targeted to basal keratinocytes, neoplastic progression occurs and is marked by an expansion of c-myc expressing basal-like cells. Expression of mad is found only in growth-arrested differentiating cells on the outer edges of preneoplastic lesions. The squamous cell carcinomas that arise evidence a variable number of sites within the tumor masses where mad expression and morphological differentiation coincide; increasing malignancy correlates with loss of both mad and capability to differentiate. These results indicate that c-Myc and Mad expression are tightly coupled to the transition from proliferation to differentiation of epithelial cells and that restriction of Mad expression may be associated with loss of normal differentiation capability and with tumorigenesis.
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Tumor suppressor loci on mouse chromosomes 9 and 16 are lost at distinct stages of tumorigenesis in a transgenic model of islet cell carcinoma.
Techniques that detect loss of genetic heterozygosity (LOH) have helped elucidate genes involved in human cancers. Previously, a genome-wide search using simple sequence length polymorphisms to detect LOH in islet cell tumors arising in a transgenic mouse model of multistage tumorigenesis had revealed two candidate tumor suppressor genes, Loh1 and Loh2, on chromosomes 9 and 16, respectively. We now have analyzed the early stages of tumor development in this model (hyperplastic, early angiogenic, and angiogenic islets) for LOH involving regions of chromosomes 9 and 16. On chromosome 9, hyperplastic and early angiogenic islets reveal a low rate of loss (< 5%) indistinguishable from background; angiogenic islets showed a 9% rate, whereas the final tumor stage had an 18% rate. By contrast, LOH was observed much earlier on chromosome 16. Notably, the LOH rate in angiogenic islets was 29%, comparable to the rate seen in end-stage tumors (32%). The results show that the two loci are lost preferentially at different stages of tumorigenesis. The observation that a high LOH rate at Loh2 is seen in the angiogenic islet stage suggests that this locus may contain an angiogenesis suppressor; in contrast, the later appearance of Loh1 may contribute to the progression from the angiogenic stage to a solid tumor. Tumors containing chromosomes with partial LOH have allowed improved localization of Loh1 to a region of approximately 3.2 centiMorgans on chromosome 9, syntenic with human chromosomes 3q and 15q.
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A transition in transcriptional activation by the glucocorticoid and retinoic acid receptors at the tumor stage of dermal fibrosarcoma development.
In transgenic mice harboring the bovine papillomavirus genome, fibrosarcomas arise along an experimentally accessible pathway in which normal dermal fibroblasts progress through two pre-neoplastic stages, mild and aggressive fibromatosis, followed by a final transition to the tumor stage. We found that the glucocorticoid receptor (GR) displays only modest transcriptional regulatory activity in cells derived from the three non-tumor stages, whereas it is highly active in fibrosarcoma cells. Upon inoculation into mice, the aggressive fibromatosis cells progress to tumor cells that have high GR activity; thus, the increased transcriptional regulatory activity of GR correlates with the cellular transition to the tumor stage. The intracellular levels of GR, as well as its hormone-dependent nuclear translocation and specific DNA binding activities, are unaltered throughout the progression. Strikingly, the low GR activity observed in the pre-neoplastic stages cannot be overcome by exogenous GR introduced by co-transfection. Moreover, comparisons of primary embryo fibroblasts and their transformed derivatives revealed a similar pattern--modest GR activity, unresponsive to overexpressed GR protein, in the normal cells was strongly increased in the transformed cells. Likewise, the retinoic acid receptor (RAR) displayed similar differential activity in the fibrosarcoma pathway. Thus, the oncogenic transformation of fibroblasts, and likely other cell types, is accompanied by a striking increase in the activities of transcriptional regulators such as GR and RAR. We suggest that normal primary cells have a heretofore unrecognized capability to limit the magnitude of induction of gene expression.
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Limited capacity for tolerization of CD4+ T cells specific for a pancreatic beta cell neo-antigen.
Mice transgenic for SV40 T antigen (Tag) under control of the rat insulin promoter (RIP) develop two alternative immunological phenotypes: tolerance or autoimmunity towards Tag. We utilized the T cell receptor (TCR) genes expressed in a Tag-specific CD4+ cell from an autoimmune RIP-Tag mouse to generate two lines of TCR transgenic mice in which either 10% or 90% of peripheral T cells express the transgenic TCR. When cross-bred to the tolerant RIP1-Tag2 line, mice from the low frequency TCR line showed partial deletion of peripheral Tag-specific T cells and nonresponsiveness of those that remained. In contrast, crossbred mice in which transgenic T cells comprised a majority of the T cell population were nontolerant both in vivo and in vitro. Thus, tolerization of CD4+ T cells specific for a rare self-antigen may fail if too many autoreactive T cells develop.
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Deregulation of both imprinted and expressed alleles of the insulin-like growth factor 2 gene during beta-cell tumorigenesis.
In a mouse model of multistage carcinogenesis elicited by the SV40 large T-antigen (Tag) oncogene in pancreatic beta cells, the gene for insulin-like growth factor IGF2 is focally up-regulated and functionally implicated in tumour development. The IGF2 gene is differentially regulated in normal tissues: the paternal allele is transiently expressed during embryogenesis, whereas the maternal allele is genomically imprinted and inactive. Crossbred mice carrying the Tag oncogene and a disruption of either the paternal or maternal allele of IGF2 reveal that both alleles are co-activated early during tumour development, and that each contributes to malignant hyperproliferation and consequent tumour volume.
Alleles↗
Vascular endothelial growth factor and its receptors, flt-1 and flk-1, are expressed in normal pancreatic islets and throughout islet cell tumorigenesis.
Endocrine organs, such as the pancreatic islets of Langerhans, contain permeable, fenestrated endothelium that allows direct access of endocrine cells to the blood stream. Factors that control differentiation and maintenance of this highly specialized endothelium remain unknown. Vascular endothelial growth factor (VEGF) is a multifunctional growth factor that may be responsible for the homeostasis of endocrine endothelium; it is a selective mitogen for endothelial cells and is able to permeabilize endothelium. We have analyzed the expression of VEGF mRNA and protein in pancreatic islet cells of normal mice and during the different stages of tumor progression in a transgenic mouse model of beta-cell carcinogenesis. The 120-amino acid and the 164-amino acid isoforms of VEGF are expressed in normal islets of Langerhans and are moderately up-regulated during the stages of tumor development. Two high-affinity receptors for VEGF, flt-1 and flk-1, are expressed by endothelial cells both in normal islets and in the stages of tumorigenesis; these receptors are not up-regulated during this process. Our data raise the possibility that VEGF is involved in the maintenance of permeable endothelium in islets of Langerhans, an observation that may have implications for islet cell physiology and diabetes. While VEGF may also play an important role in the growth of new blood vessels during islet cell tumorigenesis, it cannot be the only factor required for the activation of tumor angiogenesis.
Adenoma, Islet Cell↗
Genome-wide search for loss of heterozygosity in transgenic mouse tumors reveals candidate tumor suppressor genes on chromosomes 9 and 16.
A genome-wide scan for loss of heterozygosity (LOH) in tumors provides a powerful route to the identification of genes involved in tumorigenesis. This approach has not previously been applied to transgenic mice, despite the considerable advantages they afford for genetic dissection. Here, we report a genome-wide LOH analysis of insulinomas and carcinoid tumors in transgenic mice expressing the simian virus 40 large tumor oncogene. Although the overall genome-wide rate of LOH was quite low, chromosomes 9 and 16 showed high rates of allelic loss. About one-third of tumors showed partial LOH, allowing localization of the likely tumor suppressor genes to intervals of approximately 11 centimorgans. The locus on chromosome 9, named Loh-1, lies in a region with synteny conservation to human chromosomes 3q, 6q12, 15q24, and 3p21, while the locus on chromosome 16, named Loh-2, lies in a region corresponding to human chromosomes 3q and 22q. Of particular note is the synteny conservation with human 3p21, which shows frequent loss in human cancers. These regions do not encode two tumor suppressors, pRB and p53, known to interact with large tumor oncoprotein, suggesting the presence of new genes whose loss of function contributes to multistage tumorigenesis.
Alleles↗
T-cell tolerance toward a transgenic beta-cell antigen and transcription of endogenous pancreatic genes in thymus.
Transgenic mice expressing T antigen (Tag) in pancreatic beta cells establish systemic tolerance toward this self-protein. The self-tolerance in two families of rat insulin promoter (RIP)-Tag mice, expressing different levels of Tag protein, has been characterized. These mice have impaired antibody responses to Tag, show diminished Tag-specific T-cell proliferation, and evidence an inability to generate Tag-specific cytotoxic T cells. The existence of systemic tolerance toward a beta-cell-specific protein motivated examination of transgene expression in the thymus. Indeed, low levels of Tag mRNA were detected intrathymically. Remarkably, this expression is a valid property of the insulin gene regulatory region, since insulin RNA was also expressed in the thymus of nontransgenic mice. RNA for other pancreatic genes was also detected in the thymus, thus raising the possibility that many tissue-specific genes could be expressed intrathymically during immunological development and induction of self-tolerance. These results raise important questions for future research into the role of the thymus in tolerance induction toward so-called tissue-specific antigens.
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A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis.
Transgenic mice expressing the simian virus-40 large T-antigen (Tag) under the control of the insulin gene regulatory region offer a useful model for tumorigenesis. All the islets of Langerhans express Tag, although there is at first no aberrant proliferation. Over half of the islets become hyperplastic, however, and neovascularization of a further subset (about 10%)3 leads eventually to formation of highly vascularized solid tumours in 1-2% of islets by about 14 weeks of age. Here we show that the initial proliferative switch is correlated with focal activation of insulin-like growth factor II (IGF-II). Transfection with an antisense oligonucleotide to the IGF-II messenger RNA interferes with tumour cell proliferation in vitro, and transgenic mice homozygous for a disruption of the IGF-II gene develop tumours with reduced malignancy and a higher incidence of apoptosis. Several signals, in this case including an oncoprotein and a growth/survival factor, thus appear to be needed to elicit hyperproliferation.
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