of Squamous Carcinogenesis in the Human Cervix ... - Cancer Research

[CANCER RESEARCH57, 1294-1300, April 1. 1997]

Cross-Species

Comparison of Angiogenesis

during the Premalignant

Stages

of Squamous Carcinogenesis in the Human Cervix and K14-HPV16 Transgemc Mice1 K. Smith-McCune,2

Y-H. Zhu, D. Hanahan,

and J. Arbeit

Reproductive Endocrinology Center and Department of Obstetrics, Gynecology, and Reproductive Sciences, Box 0546 (K. S-M., Y-H. Zj, Hormone Research Institute, Box 0534 (D. H., J. Al, Department of Biochemistry and Biophysics ID. H.], and Department of Surgery (I. A.), University of California at San Francisco. San Francisco, California 94143

ABSTRACT Infection of the human cervix with certain papillomavirus subtypes is associated with the development of neoplastic squamous lesions that can progress to overt cervical malignancies. Recently, multistage squamous carcinogenesis has been achieved in K14-HPV16 transgenlc mice, wherein

expression of the human papillomavirus (IIPV) type 16 early genes Is targeted

to basal squamous

epithelial

cells by regulatory

elements of the

human keratln-14 (K14) promoter. Immunostalnlng of the endothelial marker vWf revealed a parallel upregulatlon of angiogenesis during the early neoplastlc stages in both human cervix and the epidermis of K14-

HPVI6 transgenic mice. Moreover, high-grade premslignant lesions and cancers In humans and transgenic mice were characterized by an addi tional increment In the number of new capillaries and close apposition of the micrevasculahire to the overlying neoplastic epithellum. Expression of in both species, correlating

with the increased

Although much attention has historically been paid to the angio gemc properties of tumors, in fact, recent studies demonstrate that neovascularization is also induced by premalignant lesions of the human breast, bladder, and cervix (3—7).In the uterine cervix, immu nohistochemical staining of premalignant cervical lesions for an en dothelial cell marker, vWf,3 revealed that the induction of microves sels adjacent to the basal layer of dysplastic squamous epithelium was significantly increased in high-grade as compared to low-grade le sions (6, 7). These results suggest a role for angiogenesis in the elaboration

and progression

of the premalignant

state,

prior

to the

emergence of an overtly invasive malignancy.

Observations from transgenic mouse models have added support to

the potent anglogenic factor VEGF was progressively up-regulated during carcinogenesis

study the role of angiogenesis in the development of human cervical cancer.

the hypothesis that premalignant lesions are capable of eliciting an angiogenic response. Thus, in a transgenic mouse model system of pancreatic islet (3-cell carcinogenesis, the onset of angiogenesis pre

density and

altered distribution of the microvasculature. Thus, angiogenesis occurs during the premalignant stages of squamous carcinogenesis in both hu man cervical disease and a relevant transgenlc model and may be con

ceded the development

trolled by similar molecular mechanisms in both species. These results validate the use of the transgenlc model to elucidate the role of angiogen

of malignant

tumors

(8). Moreover,

the switch

to the angiogenic phenotype presented as a focal, histologically dis tinct stage that was statistically separable both from hyperproliferation and from end-stage tumors. In a second transgenic mouse model involving the development of dermal fibrosarcomas, a potent angio genic factor, basic FGF, was found to be synthesized in normal fibroblasts, two antecedent stages of fibromatosis, and in end-stage

esis during HPV-assoclated neoplastic progression.

INTRODUCTION

It is now well establishedthat epitheial cancersproceedthrougha series of premalignant neoplastic stages prior to the emergence of an invasive malignancy. An important goal in cancer research has been the characterization of molecular genetic controls governing the tran sitions between stages during carcinogenesis. Angiogenesis is one process that appears to critically regulate the growth of established cancers (1, 2). Identification of potential mechanisms controlling angiogenesis arising during neoplastic progression in human cancers has been difficult because of restricted access to biopsies of early precursor lesions and to obvious limitations on experimental manip ulation. Transgenic models of multistage carcinogenesis have conse quently been developed to facilitate access to the premalignant stages of neoplastic growth. The research described herein compares the angiogenic properties of human cervical premalignant and malignant lesions with a murine transgenic model of squamous carcinogenesis. The purpose is to establish a relevant experimental system in which to Received 10/25/96; accepted 2/8/97.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with

18U.S.C.Section1734solelyto indicatethis fact.

fibrosarcomas.

However,

this angiogenic

factor

was released

only by

cells derived from the latter stages, which demonstrated intense an giogenesis; here again, the angiogenic switch preceded the formation of solid tumors (9). Collectively, the induction of angiogenesis in premalignant precursor lesions demonstrated in these transgemc mod els suggests that the formation of an adequate vascular network is an essential element in the maturation of premalignant lesions into their malignant counterparts. Indeed, although the angiogenic switch has conventionally been considered to be a property of malignant tumors, recent

developments

in the field have challenged

this view and have

led to an appreciation of the fact that the angiogenic switch is “turned on―in many examples of premalignant neoplastic growth (10). A clinically important example of multistage epitheial carcinogen esis is present in human cervical epitheium, wherein progressive neoplastic changes known as CIN, or cervical dysplasia, are apparent precursors to invasive cervical cancer. The designations CIN I, II, and ifi refer to mild, moderate, and severe dysplasia/carcinoma in situ, respectively. This histological grading scheme is based largely on the extent to which the thickness of the epitheium is replaced by mitot ically active cells with enlarged,

hyperchromatic

nuclei (11). A subset

of untreated dysplastic lesions, notably CIN III lesions, will advance to invasive cancers (12). Infection with HPV, found in over 90% of (NICHD) and the American Gynecological and Obstetrical Society. Support for J. M. A. wasfromgrantsfromtheAmericanCancerSocietyandtheSimonFundofthe University cervical dysplastic lesions, is strongly correlated with the develop of Californiaat San Francisco.The work performedin the Hanahanlaboratorywas mont of cervical cancer (13, 14). The molecular basis of this associ

I K. S-M. acknowledges the support ofthe Irwin Foundation, a grant from the National Cancer Institute, and the past support ofthe Reproductive Scientist Development Program

supported by grants to the National Cancer Institute, including one to D. H. and a second toJ.F.and D. H. 2 To

whom

requests

for

reprints

should

be addressed,

at Department

of

Obstetrics,

3 The

Gynecology, and Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143-0546.

abbreviations

used

are:

vWf,

von

Willebrand

factor;

CIN,

cervical

intraepitheial

neoplasia;FOF,fibroblastgrowthfactor;HPV.humanpapillomavinis;K14,keratin-l4; VEGF, vascular endotheial growth factor/vascular permeability factor.

1294

ANGIOGENE5IS IN MOUSE AND HUMAN SQUAMOUS CARCINOGENESIS

ation results from the properties of the E6 and E7 oncoproteins encoded in the early region of the “high-risk― viral genomes, such as types 16 and 18 (15). Increased understanding of the changes in cervical epithelium that contribute to cervical neoplastic progression is clearly an important goal because this disease is a world-wide health

problem

(13).

Studying

the process

in human

subjects

is,

however, difficult; thus, a representative animal model for squamous epithelial carcinogenesis is of paramount interest. Recently, a transgenic mouse model has been created in which the early region of the “high risk―viral type HPVI6 was placed under control of the human K14 enhancer/promoter (16), targeting expres sion of the early viral genes to the basal cells of squamous epithelium (17). The K14-HPV16 transgenic mice develop epidermal hyperplas tic lesions that progress to dysplastic lesions and ultimately to inva sive cancer (18—20).Because progression to squamous cell carcinoma in the epidermis of K14-HPV16 mice appeared to phenotypically parallel squamous carcinogenesis of human cervix, we have now compared the histological and angiogenic properties of both the

murine model and human disease at each stage of multistep squamous carcinogenesis. Indeed, there was a clear concordance in the histopa thology, in the patterns of vascularization, and in the expression of a potent angiogenic factor, VEGF, during carcinogenesis in both spe cies. Notably, vWf staining was markedly increased both in dysplastic

K14-HPV16 transgenic mouse skin and in CIN II and III lesions of the human cervix. A dramatic juxtaposition of the microvasculature and the overlying epithelium was also associated with advancing neoplas tic histology in both species. Marked increases in blood vessel density were accompanied by a striking incremental up-regulation of VEGF expression in squamous epithelial cells. Moreover, both up-regulation of angiogenesis and detectable VEGF expression began in the initial stages of neoplastic progression, well before the formation of invasive squamous cancer. The striking parallels between squamous carcino genesis in the Kl4-HPV16 transgenic mouse and human cervical disease offer an experimental opportunity to study both the mecha nisms coordinating angiogenesis and the contributions of neovas cularization

to the

elaboration

of multistage

squamous

epithelial

carcinogenesis. MATERIALS

AND

METHODS

Tissue Specimens, Archival human specimens were identified from the Surgical

Pathology

data base. Samples of CR4 and cancer from cervical

biopsies, loop excisions,cone biopsies, and hysterectomyspecimenswere identifiedby final diagnosison the pathologyreport.Blocks were chosen that represented the final histological diagnosis. Epidermal samples were harvested from Kl4-HPV16 transgenic mice as described previously (20) and embedded

in paraffin. Histopathological analysis was performed on H&E stained 5-pin thick sections as describedpreviously(20). Immunohistochemlcal

Staining

for vWf. Five-pin-thick

tissue sections

were cut from the paraffin block, heated for 15 mm at 60°C,deparaffinized, hydrated through graded ethanol to PBS, and incubated in 0.2% Tween 20 in

PBS for 30 rain. After rinsing in PBS, the slides were incubatedin 0.25% trypsin

and 0.025%

protease

XXIV

(Sigma

Chemical

Co.) in 0.05 M Tris-HC1

Gill's

#2 hematoxylin,

washed,

dehydrated,

and mounted

with Permount

(Fisher).

cDNA Clones, A 160-bpfragmentcorrespondingto nucleotide 145-284 of human VEGF subcloned into Bluescript H KS was a gift from the laboratory of Dr. R. Jaffe, University of California at San Francisco. A 532-bp clone of murine VEGF has been described previously (21).

In Situ Hybridization. Radioactive in situ hybridization was performed on mouse and human samples using 35S-UTP-labeled riboprobes as described previously (20, 21). To perform valid comparisons of levels of VEGF expres sion between samples, slides from each species representing each stage of the carcinogenic pathway were processed on the same day using the same probe

and were developed for equal lengths of time prior to processing. RESULTS Similar Histological Stages Are Characteristic of Multistage Squamous Carcinogenesls in Both K14-HPV16 Transgenlc Mice

and HumanCervicalDisease.Despitedifferencesin the histology of nontransgenic murine epidermis and human cervical epithelium (Fig. 1, A and E), which are based on the characteristics of keratin izing and nonkeratinizing stratified squamous epithelia, the overall organization of these two epithelia is similar. Each possesses a cu boidal innermost layer of basal cells, a transversely oriented layer of differentiating spinous cells, and upper layers of terminally differen tiated cells. These terminally differentiated cells in mouse epidermis are anucleate and contain keratohyalin granules, whereas in cervical epithelium they contain dense pyknotic nuclei but without cornified envelope formation. The earliest pathological change in the epidermis of the transgenic mouse consisted of hyperplastic lesions (Fig. 1B), which were found to be histologically analogous to human CIN I lesions (Fig. 1F). In both types of lesions, there was an expansion in the number of basal cells that dispersed throughout the innermost suprabasal layers of both epithelia. Despite this expansion of the basal cell population, the preservation of a stratified architecture in both hyperplastic mouse epidermis and human CIN I lesions indicates that the ability of the squamous epithelial cells to differentiate was preserved in these early lesions. Comparison of high-grade dysplasias and squamous cancers re vealed similarities of histopathology between the transgenic mouse model and human cervical disease (Fig. 1, C, D, G, and H). Dysplastic murine epidermal lesions (Fig. lC) and the human CIN III lesions (Fig. lG) were both characterized by the persistence of enlarged basaloid cells with irregular, hyperchromatic nuclei throughout the entire thickness of the epithelia. Mitotic figures, normally confined to the basal cell layer, were prevalent throughout the entire thickness of human cervical lesions, and immunohistochemical analysis of murine dysplasias with markers of cell proliferation confirmed the presence of mitotically active cells in the upper layers of the epidermis (17). These data are consistent with both a marked increase in proliferation and a profound disruption of the squamous epithelial differentiation program in both the murine epidermal dysplasias and the CIN Ill lesions compared to the respective hyperplastic/CIN I stages. Both

(pH 7.6) for 10 mm at 37°C, rinsedin PBS, andblockedwith 10%goat serum

murine

(VectorLabs) in PBS overnight.Slideswere then incubatedfor 2 h at room

clusters of malignant epithelial cells surrounded by fibrovascular stroma (Fig. 1, D and H). The Pattern of Angiogenesis Is Similar in Both the Transgenic

temperature in rabbit anti-human vWf serum (Dako Corp.) in PBS containing

10% goat serum and 0.05% Tween 20 (blocking solution) at dilutions of 1:6000 for human samples and 1:1250 for mouse samples. After washing in PBS-0.05% Tween 20, the slides were incubatedwith goat anti-rabbitanti serum

(Vector

Labs)

at 1:200 dilution

in blocking

solution

for 30 mm and

washed in PBS-0.05% Tween 20. After incubation in 3% hydrogen peroxide

for 30 mm, slides were incubatedin ABC Elite (Vector Labs) for 30 mm, washed

with PBS-0.05%

Tween

20, and incubated

for 5 mm in a solution

of

0.05%diaminobenzidine,0.68%imidazole,0.07%azide,and0.01%hydrogen peroxide. After three washes in water, the slides were counterstained with

Model

and human

and Human

invasive

Cervical

squamous

carcinomas

Carcinogenesis.

were composed

To compare

of

the pat

tems of angiogenesis in both transgenic mouse epidermal and human cervical lesions, the characteristics of the microvasculature in the

distinctive stages of carcinogenesis were visualized by immunohisto chemical staining for vWf. Both control (nontransgenic) mouse skin and normal human cervix contained few capillaries distributed within the dermis of the murine skin or the stroma of the cervix at a distance

1295

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Fig. 1. Histological comparisons of mouse skin and human cervical progressively neoplastic lesions. Five-pin-thick sections of mouse (A—I)) and human (E—H) specimens were stained with H&E. A, nontransgenic mouse ear, B, hyperplastic ear lesion from a K14-HPV transgenic mouse; C, dysplastic ear lesion from a K14-HPV transgenic mouse; D, poorly differentiated squamous cell carcinoma from a K14-HPV transgenic mouse; E, normal human cervix; F, human C1N I; G, human CIN ifi; H, moderately differentiated squamous cell

carcinoma. Bar: A, C, and E, 20 pin; B, D, F, G, and H, 25 pin.

from the basal cell layer (Fig. 2, A and E). Hyperplastic murine lesions

in high-grade dysplasias of both the marine epidermis (Fig. 2C) and

contained a modest increase in the number of vessels, which remained

the human cervix (Fig. 2G). New capillaries were organized into a

localized within the dermis at a distance from the affected epitheium (Fig. 2B). Similarly, the human CIN I lesions demonstrated a variable

dense microvascular array in close apposition to the overlying neo plastic epithelium. Intimate association of blood vessels and squa

increase in the number of capillaries staining for vWf in the stroma

mous epitheial cells was also evident in the invasive squamous

adjacent to the affected epithelium (Fig. 2F). In marked contrast, there was a dramatic alteration in the number and distribution of capillaries

carcinomas in both the transgenic mice (Fig. 2D) and the human cervix (Fig. 2ff). Quantification of the blood vessel density in both

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Fig. 2. Comparison of immunohistochemical staining of mouse and human progressive neoplastic lesions for vWf, an endotheial cell marker. A—D, mouse samples; E—H, human cervix. A, nontransgemc mouse ear; B, hyperplastic ear lesions from a K14-HPV16 transgenic mouse; C, dysplastic ear lesion from a Kl4-HPV16 transgenic mouse; D, poorly differentiated squamous cell carcinoma from a K14-HPV16 transgenic mouse; E, normal human cervix; F, human CIN I; G, human CIN il-ifi; H, moderately differentiated human cervical squamous cell carcinoma. A pattern of vWf staining similar to that shown in the figure was seen in 7 of 7 samples of normal human cervix (E), in 5 of 6 samples of CIN I

(F), in 4 of 4 samples of CIN u—rn, and in 9 of 10 samples of CIN Ill (G), in 3 samples of microinvasive human cervical squamous cell carcinoma, and in 7 of 10 samples of invasive human cervical cancer (H). Five of eight samples of CIN II demonstrated enhanced vWf staining with patterns ranging between those shown in F and G. Five samples of CIN H and

m did not exhibita dramaticincreasein neovascularization, but threedemonstrateda focalincrease,and two demonstrateda minimalincreasecomparedto the adjacentnormal epitheliwn. In two human cancers, the vWf staining was weaker than that shown in H but was detectable; and in one cancer, no vWf staining was apparent within the tumor. Bar, 110 pm.

1296

ANGIOGENESIS IN MOUSE AND HUMAN SQUAMOUS CARCINOGENESIS

above background in both the basal and suprabasal squamous epithe

Table duringprogressive I Increasing microvessel count and capillary-epithelialjuxtaposition squamous cers'ixAveragemicrovessel carcinogenesis of mouse skin and human

Source of sample

Range (n)

count―

Mean distance (mm/'

hal cells and in cells within the stroma (Fig. 4B). In murine epidermal hyperplasias, VEGF mRNA was just detectable above background

Range (n)

Mousec

Nontransgenic mouse

0.41 ±0.15―0.25-0.56 (2)4.67 (2)0.81

±O.16e3.0—8.0

±0.030.31—1.2 0.870.5—8.0(4)1.13

±

skin

Hyperplastic mouse

(4)3.60

skin

Dysplastic mouse

±014d0.93—1.4 (4)2.22

±0.55e0.1—6.0(4)

skin

Humai/ Normal cervix Condyloma/CIN I CIN II and ifi a The

number

of

0.475 ±0,1j98g 0.37-0.67 (7) 1.70 ±0.40 1.5—2.2 (6) 2.56 ±o.86@ 1.0—4.0 (17) discrete

vWf-stained

structures

2.95 ±0.68― 2.0-4.03 (8) 1.4 ±0.25 1.1—1.7 (6) 1.03 ±0.22― 0.72—1.5 (11)

subtending

the

epithelium

were

counted on photographs of representative fields of vWf-stained sections (mouse) or montages of the entire tissue sections (human) viewed with the X20 (mouse) or X 10

(human) objective. The length of the epithelium was measured on the photograph, and the results were expressed as microvessels per cm. Values reflect mean ±SD. b Mean

distance

represents

the

mean

of

the

distances

in

mm

between

discrete

vWf-stained structures and the basal layer of the epithelium, as measured on photographs described above. For each histological

sample, 13—18measurements

were made and

(Fig. 3D), whereas in human CIN I lesions, there was an additional increment in VEGF mRNA expression compared to normal cervical epithelium (Fig. 4D); in both species, the levels were increased above those of the normal samples. The level and pattern of VEGF mRNA expression was also similar in high-grade squamous epithelial dysplasias in both mouse and human (Figs. 3F and 4F). The marked increase in the VEGF signal was localized within the dysplastic keratinocytes of the mouse skin and the human cervix. A further increase in VEGF expression was apparent in both the well-differentiated and moderate to poorly dif ferentiated transgenic murine squamous carcinomas (Fig. 3, H and 3). VEGF mRNA was also detected in human invasive cervical cancer (Fig. 4H). A quantitative analysis of VEGF mRNA expression at each stage of cervical carcinogenesis has been reported recently by another group

averaged,andtheaveragesfromeachsamplewereusedto calculatethemeanandSDfor

(7). Our data demonstrate a parallel pattern in the murine model of

each stage of histology.

cervix were obtained from hysterectomy specimens of patients with benign cervical

squamous carcinogenesis. Collectively, these data demonstrate that induction of VEGF mRNA is an early event in squamous epithelial carcinogenesis. Moreover, the close correlation of incremental VEGF expression with the formation and localization of new capillaries suggests that increasing levels of this angiogenic regulatory factor

histology. Samples of condyloma and CIN were obtained from loop and cone biopsy

contributes

C Samples

were

obtained

from

HPVI6-K14

transgenic

mice

as described

in “Materials

and Methods―. d p

0.023

ep

Ø@5 bytwo-sample t test(two-tailed) comparing normal todysplasia.

by two-sample

t test (two-tailed)

comparing

normal

to dysplasia.

I Human samples were obtained from the pathology archives. Samples of normal

specimens. S p = 2 X lO@ by two-sample t test (two-tailed) comparing normal to CIN II and Ill. h p

o.ooos

by two-sample

t test (two-tailed)

comparing

normal

to CIN

II and

to the regulation

of neovascularization

in both species.

III.

DISCUSSION mouse and human lesions at each stage of neoplastic progression revealed a statistically significant increase in the microvessel count from normal to dysplasia in both species (Table 1). A similar increase has been reported previously for human lesions (6, 7). However, these studies

used

histologically

normal

cervical

epithelium

adjacent

to

dysplastic epithelium as the normal samples. We have extended those studies by using, as the control samples, slides of normal cervix from patients with no evidence of HPV-related disease. To quantitate the observation that the microvasculature associated with high-grade dysplasias appeared to be closer to the overlying epithelium than that associated with normal epithelium, we have measured the distances between discrete vWf-stained structures and the overlying basal cell layer for mouse and human premalignant samples. Indeed, there was a statistically significant decrease in the mean distance between the microvasculature and the basal cell layer of the epithelium comparing normal to high-grade dysplasias in both species (Table 1). Thus, the epithelial angiogenic response elicited by these squamous neoplasias appears to possess two components: an intense upregulation of angiogenesis; and a dramatic juxtaposition of the neovasculature to high-grade premalignant lesions. It is apparent that these two facets of the angiogenic response occur at similar stages of neoplastic progression in both the transgenic mouse model and the human cervix. Incremental Up-Regulation of VEGF in Squamous Carcinogen esis in Both Species. To begin an investigation into the mechanisms underlying the observed up-regulation of angiogenesis in squamous

A consistent feature of squamous carcinogenesis in both the murine Kl4-HPV16 transgenic model and human cervical lesions is its mul tistage nature. The results presented here indicate that histologically similar lesions from the two species demonstrate parallel progression from intraepithelial premalignant stages to invasive cancer. The cor relation is clear and consistent despite the fact that the lesions in the transgenic mouse model develop in a cornified epidermis, whereas human CIN lesions develop in a mucosal squamous epithelium. Our data demonstrate that angiogenesis is an early component of squa mous multistage carcinogenesis in both species. Historically, the angiogenic switch was thought to occur concomitant with the appear ance of malignant tumors. Recent data have challenged that hypoth

esis (10). Indeed, our results reveal that the onset of angiogenesis

occurs very early during the premalignant stages in two different examples of epithelial carcinogenesis. The data suggest that the proc ess of neovascularization in premalignant epithelial neoplasias is composed of two distinct stages. Initially, it is characterized by a modest increase in the number of microvessels apparent in the stroma or dermis underlying the epithelium in the early phases of neoplastic growth. A second component of epithelial angiogenesis, characterized by close apposition of the microvasculature to the neoplastic lesions in high-grade dysplasias, is quantitatively analyzed in this report. The pronounced juxtaposition of vessels to the basal cell layer in advanced dysplasias suggests a powerful chemoauraction of the neovasculature to the dysplastic epithelium, perhaps in response to cytokines and carcinogenesis, the expression of VEGF mRNA was analyzed by in growth factors produced by the neoplastic cells. In this regard, it is interesting that VEOF has recently been demonstrated to have che situ hybridization and compared at each stage of mouse and human motactic properties for endothelial cells (22). Notably, the two facets squamous epithelial carcinogenesis. There were both distinctive dif ferences and striking similarities in the pattern of VEGF expression in of angiogenesis occur at parallel stages of neoplastic progression in both the Kl4-HPV16 transgenic mouse and the human cervix. It will the murine epidermal and human cervical multistage pathways (Figs. 3 and4). As shownin Fig. 3B, VEGF expression wasnotdetectable be interesting to determine whether the parallels described here be in either the dermis or the epidermis of nontransgenic mouse skin. In tween the mouse and human systems reflect common underlying physiological effects of the HPV oncoproteins or general mechanisms contrast, the human cervix contained VEGF mRNA at a low level just

1297

ANGIOGENESIS IN MOUSE AND HUMAN SQUAMOUS CARCINOGENESIS

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Fig. 3. Detection of VEOF mRNA by in situ hybridization of progressive epithelial neoplastic lesions from the K14-HPV transgenic mouse. A and B, nontransgenic mouse skin;

C and D, hyperplastic transgenic mouse skin; E and F, dysplastic transgenic mouse skin; G and H, well-differentiated squamous cell carcinoma from a transgenic mouse; I and J,

moderatelydifferentiatedsquamouscellcarcinomafroma transgenicmouse.Brightfield(A,C. E. G, andI) anddarkfield(B,D, F, H, andJ) photographsof mouseskinsamples after in situ hybridization with 35S-UTP-labeled riboprobes as described previously (20, 21). Samples of tissue from three individual mice representing each stage were prepared and

analyzed in parallel, and the photographs of representative fields are shown. Bar: A—F, 25 pin; Cl—i, 50 pm.

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cervix; C and D, CIN I; E and F, CR4 H-ffl; G and H, invasive cervical squamous cell carcinoma. Bright field (A, C, E, and G) and dark field (B, D, F, and H) photographs of human hysterectomy (A, B, G and H) and loop (C—F) specimens after in situ

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governing epithelial carcinogenesis independent of the presence of HPV. However, the parallels are striking for the appearance of the angiogenic switch prior to the development of invasive cancer. VEGF, one of the most common angiogenic factors expressed in human malignancies, has been shown to be required for sustaining angiogenesis and consequent growth of established cancers in murine transplantation studies (23, 24). For example, innoculation with neu

tralizing antibodies to VEGF can impair growth of human glioblas tomas and human colon carcinoma in immunodeficient mice (25, 26). In addition, a dominant-negative mutation of a VEOF receptor can result in inhibition

of glioblastoma

tumor

growth

in vivo (27). Thus,

interference with an inducer of angiogenesis can inhibit neovascular

ization and thereby constrain malignant growth. The data presented here demonstrate a progressive up-regulation of a potent angiogenic

factor, VEGF, in the premalignant stages and in the squamous carci nomas of mouse skin from the K14-HPV16 mouse. Guidi et a!. (7) have recently quantitatively analyzed VEGF up-regulation during

cervical carcinogenesis

in humans; in that report, the authors found

significantly greater expression of VEGF by in situ hybridization in high-grade dysplasias compared to histologically benign squamous epitheium. Our data confirm their findings and demonstrate a parallel phenomenon in a transgenic mouse model. The human cervix and the epidermis of the HPV16-Kl4 transgenic mouse constitute the first examples in which the onset of angiogenesis and induction of VEGF are clearly observed in the premalignant stages of squamous carcino genesis. In contrast, in a multistage pathway to islet cell carcinomas of another transgenic mouse model, VEGF was found to be expressed in normal pancreatic islets and throughout tumorigenesis (21). This constitutive VEGF expression in normal pancreatic islets, in contrast to the lack of detectable VEGF mRNA in normal squamous epithe hum of the Kl4-HPV16 transgenic mice, suggests that VEGF regu lation is cell type specific, and that angiogenesis accompanying tu mongenesis may be under distinct molecular controls in different tissues.

1299

ANGIOGENESIS

IN MOUSE AND HUMAN SQUAMOUS

It is known that VEGF expression is regulated by metabolic con ditions such as hypoxia and hypoglycemia (28—30)and by certain oncogenes, including activated H-ras (31, 32). Analysis of the induc tion of VEGF expression and the localization of VEGF mRNA within the hyperplastic and dysplastic murine epidermis and human cervix implicates both HPV oncogene expression and hypoxia as possible mediators of VEGF upregulation. A contribution of the HPV onco proteins to VEGF up-regulation is suggested by the similar pattern of incremental upregulation both of the HPV oncogenes and VEGF mRNA in the progression from hyperplasia to dysplasia and invasive carcinomas in the mouse transgenic model (Fig. 3 and Ref. 18). In addition, it is notable that the signal for VEGF mRNA detected by in situ hybridization

in neoplastic

transgenic

epidermis

and derivative

squamous cancers was most intense in the interior of lesions and at the tips of papillary fronds; these are regions in which the perfusion

gradient and tissue 02 tension might be expected to be low, resulting in metabolic conditions known to cause VEGF mRNA up-regulation. The contribution of signaling via additional growth factors to squamous epithelial angiogenesis must also be considered. Thus, we have recently observed progressive up-regulation of FGF-l, FGF-2, and their receptors in the dysplastic and malignant phases of neoplasia and squamous cancer in K14-HPV16 transgenic mice, suggesting that angiogenesis may be combinatorially regulated at distinctive stages of squamous carcinogenesis (33). The possible roles of these and other angiogenic factors in the human cervical pathway, as well as the roles of angiogenic inhibitors in both human cervix and mouse epidermis, remain to be assessed. In conclusion, the results presented here demonstrate close parallels between

progressive

squamous

carcinogenesis

and neovascularization

in the human cervix and the epidermis of Kl4-HPV16 transgenic mice. The role of premalignant angiogenesis in determining the ulti mate fate of dysplastic lesions is largely unexplored. The transgenic model, therefore, provides the opportunity to experimentally manip ulate the microvasculature during tumor development, both to assess the functional significance of the early activation of angiogenesis and as a platform for preclinical testing of therapies. Thus, for example, genetic complementation tests can be performed by cross-breeding the

K14-HPV16 mice to other transgenic mice to assess the functional contributions of candidate effectors such as VEGF. A parallel strategy could use the transgenic mice for therapeutic trials to determine the

antineoplastic effect of pharmacological abrogation of the angiogenic

CARCINOGENESIS

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ACKNOWLEDGMENTS We acknowledge support for equipment by the Markey Charitable Trust to the University of California at San Francisco Transgenic Mouse Facility. The authors thank P. Bacchetti in the Department ofBiostatistics and Epidemiology for assistance with statistical analysis and J. M. McCune, S. Shivakumar, and

R. Wiener for criticalreadingof the manuscript.

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