Profiling Microdissected Epithelium and Stroma to Model Genomic Signatures for Cervical Carcinogenesis Accommodating for Covariates

Research Article

David Gius,1 Margo C. Funk,2 Eric Y. Chuang,1 Sheng Feng,3 Phyllis C. Huettner,4 Loan Nguyen,2 C. Matthew Bradbury,1 Mark Mishra,1 Shuping Gao,1 Barbara M. Buttin,2 David E. Cohn,2 Matthew A. Powell,2 Neil S. Horowitz,2 Bradford P. Whitcomb,2 and JanetS. Rader 2

1Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and 2Division of Gynecologic Oncology, Department of Obstetrics and Gynecology; 3Division of Biostatistics; and 4Lauren V. Ackerman Laboratory of Surgical Pathology, Washington University School of Medicine, St. Louis, Missouri

Abstract (CIN 1–3) and finally to squamous cell carcinoma antigen (SCCA) This study is the first comprehensive, integrated approach to have been well characterized. Histologically, CIN 1 consists of examine grade-specific changes in gene expression along the immature basal-type cells involving the lower third of the entire neoplastic spectrum of cervical intraepithelial neoplasia epithelium. In CIN 2, these immature basal-type cells involve more (CIN) in the process of cervical carcinogenesis. This was than the lower third, whereas CIN 3 involves the full thickness of the accomplished by identifying gene expression signatures of epithelium. In addition, higher CIN grades exhibit nuclear crowding, disease progression using cDNA microarrays to analyze RNA pleomorphism, loss of cell polarity, and increased mitotic activity from laser-captured microdissected epithelium and underlying (1). These transitions seemto be well conserved and, as such, stroma from normal cervix, graded CINs, cancer, and patient- provide an intriguing systemto use genomicsto identify the early matched normal cervical tissues. A separate set of samples were events in cervical cell transformation. The underlying stromal subsequently validated using a linear mixed model that is ideal composition may vary slightly beneath the basement membrane; to control for interpatient gene expression profile variation, however, it is overwhelmingly characterized by fibroblasts. Complex such as age and race. These validated genes were ultimately molecular cross-talk between the epithelium and stroma has been used to propose a genomically based model of the early events implicated as a key component of neoplastic transformation, tumor in cervical neoplastic transformation. In this model, the CIN 1 invasion, and metastasis. Therefore, to construct an effective model transition coincides with a proproliferative/immunosuppres- for CIN progression in vivo, it is imperative to separately examine sion gene signature in the epithelium that probably represents the different microenvironments involved in carcinogenesis. the epithelial response to human papillomavirus infection. The Multidisciplinary studies have unequivocally implicated human CIN 2 transition coincides with a proangiogenic signature, papillomavirus (HPV) as the etiologic agent of neoplastic cervical suggesting a cooperative signaling interaction between stroma lesions, with HPV DNA being found in 99.7% of invasive cervical and tumor cells. Finally, the CIN 3 and squamous cell carcinoma carcinomas (2). Even so, most HPV infections are transient and antigen transition coincide with a proinvasive gene signature spontaneously cleared by the host immune system. Thus, few that may be a response to epithelial tumor cell overcrowding. women infected with HPV develop CIN 3 and fewer still progress This work strongly suggests that premalignant cells experience to invasive carcinoma (3), suggesting that HPV does not act alone a series of microenvironmental stresses at the epithelium/ in the development of cervical cancer. Persistence of HPV infec- stroma cell interface that must be overcome to progress into a tion and progression to cancer vary by age and race: Women over transformed phenotype and identifies the order of these events 30 years show a much higher persistence of HPV than younger in vivo and their association with specific CIN transitions. women (4, 5), and progression to CIN 3 may vary by race (6). [Cancer Res 2007;67(15):7113–23] To address interpatient variability, tissue heterogeneity, and experimental reproducibility, we used laser-capture microdissection Introduction (LCM) on 130 prospectively collected samples to isolate epithelial cells and stromal fibroblasts within 3 mm of the basement Cervical malignancies present a unique opportunity to profile membrane from normal cervix, graded CINs, cancer, and patient neoplastic transformation. The histologic transition from normal compartment–matched ‘‘normal’’ cervical tissues. Microarray epithelial cells to preinvasive cervical intraepithelial neoplasia hybridization was done using only excellent quality RNA with triplicate replication per sample and a single universal reference

Note: Supplementary data for this article are available at Cancer Research Online across all samples. We identified specific markers in the epithelium (http://cancerres.aacrjournals.org/). and stromal compartments showing a progressive change in gene D. Gius and M.C. Funk contributed equally to this work. expression (increased or decreased) fromviral cytopathic to inva- Current address for E.Y. Chuang: Biomedical Engineering Group, Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan. sive cancer and markers with a sharp change at CIN 3 and inva- Requests for reprints: David Gius, Radiation Oncology Branch, National Cancer sive cancer. Validation on a separate set of samples was improved Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-496-5457; Fax: 301- by using a linear mixed model accommodating for age and race 480-5439; E-mail: [email protected] and Janet S. Rader, Departments of Obstetrics and Gynecology and Genetics, Washington University School of Medicine, 4911 and showed several intriguing findings. Both preneoplastic epithe- Barnes-Jewish Hospital Plaza Box 8064, St. Louis, MO 63110. Phone: 314-362-3181; Fax: lial cells and the surrounding fibroblasts induce the expression 314-362-2893; E-mail: [email protected]. I2007 American Association for Cancer Research. of proangiogenic factors, suggesting a potential cooperative com- doi:10.1158/0008-5472.CAN-07-0260 munication between these differing cell types at the transition www.aacrjournals.org 7113 Cancer Res 2007; 67: (15). August 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research through CIN 2. In addition, distinct functional gene expression the indirect patient-matched abnormal/normal expression ratio that was profiles were identified as cells progressed into the different CIN and used in all downstreamdata analyses. SCCA subtypes. The in vivo model shows three distinct genomic Semiquantitative and real-time PCR. Reverse transcription was done signatures at the epithelium-stromal interface: (a) early viral/CIN 1 on each aRNA sample used to confirm the microarray results. Using the RETROscript First Strand Synthesis kit (Ambion) and random decamers, proproliferative/immunosuppression signature in the epithelium; we reverse-transcribed 2.5 Ag of each aRNA sample according to the (b) intermediate CIN 2 proangiogenic epithelium and stromal manufacturer’s instructions. The resulting cDNA was semiquantitatively signature; and (c) late CIN 3/SCCA proinvasive epithelial and amplified in 25 to 30 cycles, using primer sets specifically designed for the stromal signature. 3¶-biased products of the T7-based RNA amplification. Online supplemental material. Supplemental Data includes the characterization of the patients validation set (Supplementary Table S1); Materials and Methods a detailed description of analyses I and II; and additional details for the Tissue collection, processing, slide preparation, and LCM. This study Materials and Methods for tissue processing, RNA isolation and amplifi- was approved by Washington University Human Studies Committee, and cation, and HPV typing. informed consent was obtained from all patients. Specimens excised from the cervix were placed in optimal cutting temperature embedding medium Results for frozen tissue specimens (Sakura Finetek USA). Approximately fifty 8-Am sections were cut per block. A reference slide was prepared at every 10th LCM and preparation of samples of epithelium and under- section and examined by a gynecologic pathologist (P.C.H.) who marked the lying stroma from sequential neoplastic grades. To investigate exact location in the epitheliumand stromato be dissected and were the changes in genomic expression that occur as normal cervical validated in the LCM cap. The epitheliumwas more than 85% epithelial cells tissue progresses to CIN and then to cervical carcinoma in specific and the stroma was more than 85% fibroblasts. The mean depth of dissection microenvironments, we used LCM (Supplementary Figs. S1A–C)to was 2 mm (1–3 mm). For further details, see Supplementary Data. isolate the epithelium and stromal compartments from the same RNA isolation and amplification. Immediately after LCM, each cap area of cervix fromeach neoplastic grade. Dissected samplesfrom A was placed in a 0.5 mL Eppendorf safe-lock tube containing 30 Lof the epitheliumcontained morethan 85% epithelial cells and the extraction buffer fromthe PicoPure RNA Isolation Kit (Arcturus; see stroma over 85% fibroblasts. The maximum height of the Supplementary Data). RNA quality were assessed using the Nanodrop Spectrophotometer (Nanodrop Technologies) and RNA 6000 Pico LabChip epithelium was 3 mm from the basement membrane with most Kit in conjunction with the Agilent 2100 Bioanalyzer (Agilent Technologies), samples being 1 to 2 mm. The stromal maximal dissection was less respectively. Only high-quality RNA with an 18S:28S rRNA ratio >1.0 was than or equal in height to the epithelium, usually 2 to 3 mm. The applied to the microarrays. An input of 10 to 20 ng of total cellular RNA basement membrane was included in the epithelium. One hundred containing poly(I)Ápoly(C) (Amersham Biosciences) as a nucleic acid carrier and thirty samples were meticulously marked for dissection by an was amplified in two rounds of T7-based in vitro transcription, using the experienced gynecologic pathologist (P.C.H.). This is critically RiboAmp HS Amplification Kit (Arcturus) according to the manufacturer’s important to this work because the proper histologic categories instructions. A universal reference RNA (Stratagene) was in all microarray are necessary to produce the model of gene expression changes. hybridizations. Patient-matched neoplastic and normal epithelial samples were Microdissected specimens. A total of 133 RNA samples were amplified microdissected from five disease-free histologically normal speci- from microdissected epithelium/tumor (86 samples) and stroma (47 samples). Represented in this total were 85 samples for microarray hybridization, mens, three viral cytopathic effect (VCE), five CIN 1, four CIN 2, including 26 epithelial and 9 stromal abnormal-normal patient matched seven CIN 3, and six squamous cell carcinoma human cervical pairs (52 and 18 samples, respectively) and 10 epithelial and 5 stromal samples specimens. Patient-matched neoplastic and normal stromal samples from normal transformation zone. A validation set of 21 epithelial and 24 were microdissected from two disease-free histologically normal stromal samples ranging from normal tissue to cancer were obtained, micro- specimens, two CIN 1, three CIN 2, two CIN 3, and two squamous dissected, and processed in the same way as the discovery panel (shown in cell carcinoma specimens. Patient characteristics include age, race, detail in Supplementary Table S1). and HPV status (Table 2; Supplementary Table S1). A validation set Microarray fabrication, hybridization, and analysis. The microarrays of 21 epithelial and 24 stromal samples ranging from normal tissue used for this study (National Cancer Institute ROSP 8 k human array) were to cancer were obtained, microdissected, and processed in the same prepared fromthe Research Genetics NamedGenes set and contained 7,680 way as the discovery panel (Table 2; Supplementary Table S1). human cDNA clones enriched for known genes. cDNAs were spotted onto RNA was extracted fromthese samplesand analyzed on cDNA poly-L-lysine–coated slides using an OmniGrid arrayer (GeneMachines), as previously described (7). Probe labeling, microarray hybridization, and microarrays. To ensure accurate hybridization, we used only RNA scanning were done as previously described (8) with few modifications. For samples with a 28S:18S ratio >1.0 (Supplementary Fig. S1D). Even all experiments, the cDNA probes from microdissected abnormal and with the most rigorous technique, such high-quality RNA was normal specimens were compared with an amplified universal reference obtained less often fromstromathan fromepithelium;thus, RNA (Stratagene). For each array, 3 Ag of amplified RNA (aRNA) from a slightly fewer stromal samples were analyzed on the microarrays. single microdissected sample and 6 Ag of universal reference aRNA were To obtain enough RNA fromLCM, we did two rounds of T7 linear labeled with Cy3-dUTP and Cy5-dUTP, respectively. Microarrays were amplification on all samples (Supplementary Fig. S1E). aRNA was A scanned at 10 mresolution, using a GenePix 4000A scanner (Axon then hybridized to cDNA microarrays against an amplified Instruments, Inc.). The hybridized Cy3- and Cy5-labeled cDNA samples were universal reference RNA in triplicate. After a pilot study in our scanned at 532 and 635 nm, respectively, and the resulting TIFF images were 5 analyzed with GenePix Pro 3.0 software (Axon Instruments). laboratory showed that indirect (abnormal or normal to reference) Data processing. Array elements that were flagged or had a median and direct (abnormal to normal) hybridization gave similar results, signal/background intensity <1.4 were excluded fromfurther analysis. we decided to use indirect hybridization, which provides more Global normalization of median signal and background intensities was done flexibility for data analysis. To avoid dye bias, we labeled all the before background was subtracted fromlocal signal intensities. The mean Cy3/Cy5 ratio of 3 hybridization replicates representing abnormal/reference was divided by the mean Cy3/Cy5 ratio of normal/reference to determine 5 Unpublished data.

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Figure 1. Differential expression along the progression from normal cervical tissue to cancer in abnormal versus patient-matched normal microdissected cervical specimens from microarray. A, differentially expressed genes in the epithelium. B, differentially expressed genes in stroma. Graphed values represent mean age- and race-adjusted intensities for each histopathologic grade. Black line, mean expression for histologically normal samples. Red and blue lines, increasing or decreasing expression, respectively. Colored boxes, different histologic grades. Y axis, the microarray fold change. experimental samples with the same dye (Cy3) and always labeled and abnormal samples. To best identify the significant genes the reference RNA with Cy5. To achieve the statistical power and corresponding to disease progression in human samples, two correct for interpatient and experimental variation, 246 cDNA different analysis methods were used. For analysis I, we used microarrays and more than 50 quality control microarrays were Functional Genomics v7.2 (Spotfire DecisionSite) to identify (a) used to evaluate LCM samples. The raw data for this analysis can increasing or decreasing abnormal/normal gene expression ratios, be obtained online. and (b) abrupt changes in gene expression between two CIN grades. Analysis of epithelium and stromal samples from each For analysis II, we generated a mixed linear model to fit the neoplastic grade. We identified continuous trends in gene experimental design structure and accommodate multiple cova- expression along the progression fromnormalcervical tissue to riates. We identified variables and patterns that could be factored cancer and compared these trends in patient-matched normal into the model to reduce interpatient variation and eliminate some www.aacrjournals.org 7115 Cancer Res 2007; 67: (15). August 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research possible false positives and therefore improve the pathway model. did not change as disease progressed (Fig. 1; Supplementary The false discovery rate was calculated using the multitest procedure Table S2; Lists 2, 4, 6, 8, 10, 12, 14, and 16), whereas in pattern 2 in SAS by Benjamini and Hochberg (SAS Institute; ref. 9). Several (parallel), expression of target genes changed in both normal interesting patterns were identified, modeling increasing and (immediately adjacent to abnormal) and neoplastic tissue with decreasing gene expression over disease progression and extent of disease progression (Fig. 1; Supplementary Table S2; Lists 1, 3, 5, 7, gene expression within an individual neoplastic grade. In pattern 1 9, 11, 13, and 15). Pattern 1 identifies genes in neoplastic (nonparallel), gene expression in the adjacent normal specimens mechanisms whereby gene expression changes within histologic

Figure 2. Semiquantitative RT-PCR validation of selected genes from cervical epithelium and stroma. A, 21 microdissected epithelium. B, 24 microdissected stroma. Smaller P values represent the combination of stronger upward or downward gene expression trends across the neoplastic progression and smaller degrees of variance between values of the same neoplastic grade. Graphed values represent the percentage of maximum gene intensity/glyceraldehyde-3-phosphate dehydrogenase (Y axis; age- and race-adjusted intensities) for each histopathologic grade. The X axis is labeled with the corresponding histologic grade.

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Figure 3. Schematic model for cervical carcinogenesis. Significant expression patterns using validated genes from analyses I and II are shown beneath the histologic grades; VCE, CIN 1 to 3, and SCCA. Yellow and red horizontal lines, basement membrane that separates the epithelium and stromal compartments. Genes coded in dark red, validated genes with increased expression; genes in dark blue, validated genes with decreased expression. Additional genes are also depicted in the model: gray, previously identified host and viral genes; light red or light blue, genes that increase or decrease, respectively, form the microarray data but are not yet validated. The CIN 1 transition coincides with a proproliferative/immunosuppression gene signature that appears to represent the epithelial response to HPV infection. The CIN 2 transition coincides with a proangiogenic stroma/epithelium signature, suggesting a cooperative signaling interaction between stromal and neoplastic cells responding to a shortage of nutrients necessary for proliferation. The CIN 3/SCCA transition coincides with a proinvasive gene signature that may be responding to tumor cell overcrowding. progression, whereas pattern 2 identifies genes in which the normal environmental cofactors. Seven genes initially validated from adjacent epitheliumor stromaparallels the expression pattern of analysis I failed to appear at the top of the 16 gene lists from the neoplastic area and denote more globally changes occurring in analysis II. However, analysis II provided a list of genes that showed the diseased cervix. These analytic methods also identified genes more consistent expression trends across cervical neoplasia in the that changed their expression sharply at the threshold between CIN two populations of cervical samples. The expression level from 3 and cancer and might therefore be markers of invasion (Fig. 1; normal to cancer for the significant genes changed by a factor of Supplementary Table S2; Lists 5–8 and 13–16). 1.8 to 2.4 (Fig. 2). To compare the results across methods, we prepared a second Epithelium and stromal gene expression patterns identify population of microdissected epithelium and stroma cells for unique CIN progression pathways. The expression pattern of validation (Supplementary Table S1). We did semiquantitative significant genes showed identical trends through the histologic reverse transcription-PCR (RT-PCR) on select genes fromthe top of grades of cervical carcinogenesis for both the discovery and each list obtaining <50% validation for analysis I and 68% (27 of 40) validation sample set. For example, analysis II identified CENPF, for analysis II. Validation required the identical trend in gene CDKN2A, ITGAV, and KIF23 as showing nonparallel expression expression fromnormalto cancer as identified fromthe array data (Fig. 1). In contrast, MAPK7 and HINT1 are representative of genes (Figs. 2 and 3; Table 1). Smaller P values represent the combination showing parallel expression; the stroma beneath morphologically of (a) stronger upward or downward gene expression trends across normal epithelium parallels the expression of the stroma beneath the neoplastic progression and (b) smaller degrees of variance the adjacent neoplastic epithelium(Fig. 1 B). IFNAR1 (Fig. 1A) was between values of the same neoplastic grade. This validation rate is identified in analysis II as a sharp nonparallel increase in the high considering the genetic variability between individuals and epithelium. Visual inspection shows that IFNAR1 actually begins to www.aacrjournals.org 7117 Cancer Res 2007; 67: (15). August 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research increase in the neoplastic and adjacent normal epithelium at VRE to that of CDKN2A, which is undergoing extensive clinical testing as but shows divergent and nonparallel expression at CIN 3. Overall, a cervical cancer screening marker. The raw data and gene lists there was much less variability between the stroma underlying (Table 3) provided here will provide a framework for the normal and neoplastic epithelium then within the neoplastic establishment of an evolving model of cervical carcinogenesis at epitheliumand adjacent normaland significant gene expression the epithelium-stromal interface and biomarker development. changes in the stromal fibroblasts did not begin until CIN 2. Evaluation of HPV status in microarray samples. HPV status These expression patterns can also be seen in the validation set. was characterized along the neoplastic progression by using general Microdissected histologically normal epithelial cell samples from primer PCR, denaturing high-performance liquid chromatography, patients with cancer (graphs 1 and 2, Fig. 2) show differential and sequencing for each specimen (Table 2; ref. 10). In epithelium, expression patterns paralleling neoplastic epitheliumor stromawhen low-grade cervical lesions (VCE and CIN 1) were characterized by a compared with histologically normal samples from patients with mixture of low-risk (HPV 53, 84), intermediate-risk (HPV 39, 52, 56), no cervical pathology (graphs 3–8, Figs. 1 and 2) for IFNAR1, EMP1, and high-risk (HPV 16, 18, 31, 45) types. High-grade dysplasia (CIN 2 IL1RN, ACCA1,andMMP3. MMP3 was initially identified by its sharp and 3) was characterized primarily by high-risk HPV (HPV 16, 18, increase in expression at the threshold between CIN 3 and cancer in 31, 59) and a few intermediate-risk HPV types (HPV 52, 58). the stroma and epithelium (Fig. 1) and then validated (Fig. 2A and B). Histopathologically confirmed squamous carcinoma cells contained We show that microdissection of epithelium and stroma can identify only high-risk HPV types (HPV 16, 18, 31, and 45). There was a genes showing opposite trends in expression. DSG3 shows decreasing complete absence of HPV DNA from all disease-free cervical expression in the epitheliumand increasing expression in the stroma specimens. A few specimens contained more than one HPV type. through disease progression (Figs. 1 and 2). To determine whether our approach can identify potential clinical screening markers, we did semiquantitative RT-PCR for KIF23, Discussion using RNA from a third population of Pap smears (Supplementary This study is the first comprehensive, integrated approach to Fig. S2). This gene has not previously been identified as a marker examining histologic, grade-specific changes in gene expression of carcinogenesis, but its expression more than doubled in the along the entire neoplastic spectrumof cervical carcinogenesis. microarray and validation studies (Figs. 1 and 2). It was also This study is also the first to use LCM to isolate and describe the overexpressed in ‘‘normal’’ cervical epitheliumfromcancer patients distinct cell populations of the epithelial and stromal micro- in the validation set, its nonparallel expression pattern being similar environments and to use patient-matched, histopathologically

Table 1. Differentially expressed genes from microarray analyses I and II

Gene Microenvironment Histopathologic Expression pattern Array P grade of first expression change

CENPF EpitheliumVCE Increasing, not parallel; increase at cancer P = 0.001; P = 0.00001 CDKN2A EpitheliumVCE Increasing, not parallel; increase at cancer P = 0.001; P = 0.00001 ITGAV EpitheliumVCE Increasing, not parallel; increase at cancer P = 0.00003; P = 0.00472 KIF23 EpitheliumVCE Increasing, not parallel; increase at cancer P = 0.00099; P = 0.00049 IFNAR1 EpitheliumVCE Increasing; increase at cancer Visual inspection; P = 0.00288 EMP1 EpitheliumCIN 1 Decreasing, not parallel; decrease at cancer P = 0.040; P = 0.00051 IL1RN EpitheliumCIN 1 Decreasing, not parallel; decrease at cancer P = 0.027; P = 0.012 ACAA1 EpitheliumCIN 1 Decreasing, not parallel; decrease at cancer P = 0.035; P = 0.00086 KAL1 EpitheliumCIN 2 Increasing, not parallel P = 0.00075 CDK4 EpitheliumCIN 2 Increase cancer (analysis I) P = 0.108; P = 0.050 MMP3 EpitheliumCIN 3 Increasing, not parallel; increase at cancer P = 0.00078; P = 0.469 TLOC1 EpitheliumSCCA Increasing parallel, not parallel; increase P < 0.00001; P = 0.224 at cancer MMP14 EpitheliumSCCA Increasing, not parallel; increase at cancer P = 0.030; P = 0.518 DSG3 EpitheliumCIN 3 Decreasing, not parallel; decrease at cancer; P = 0.175; P = 0.075 (P = 0.038) (analysis I) NOMO2 EpitheliumSCCA Increase at cancer (analysis I) P = 0.504; (P = 0006) TNFRSF12A EpitheliumSCCA Increase at cancer (analysis I) P = 0.701; (0.00061) TBX19 Stroma CIN 2 Decreasing, not parallel P = 0.02 DAB2 Stroma CIN 2 Decreasing, parallel P = 0.00096 MAP2K7 Stroma CIN 2 Decreasing, parallel P = 0.00031 HINT1 Stroma CIN 2 Increasing, parallel P = 0.006 TAGLN2 Stroma CIN 2 Increasing, parallel P = 0.00005 DSG3 Stroma CIN 3 Increasing, not parallel; increase at cancer P = 0.428; P = 0.298; (P = 0.029) (analysis I) BRWD1 Stroma SCCA Increasing, not parallel; increase at cancer P = 0.002; P = 0.744 MMP3 Stroma SCCA Increasing, not parallel; increase at cancer; P = 0.208 P = 0.89467 (P = 0.062) (analysis I)

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Table 2. Patient characteristics and HPV typing for microarray specimens

Age (y) Race Histopathology Epithelium Stroma

Sample ID HPV type Sample ID HPV type

Abnormal Normal Abnormal Normal

57 Black Normal cervix 227 NA None 228 NA None 49 White Normal cervix 191 NA None 192 NA None 32 Black Normal cervix 169 NA None * 72 White Normal cervix 205 NA None * 39 White Normal cervix 209 NA None * 47 White Normal cervix * 226 NA None 20 White VCE/matched normal 118/119 84 84 * 23 Black VCE/matched normal 155/156 31 None * c 45 Black VCE/matched normal 214/218 None 18 * 18 Black CIN 1/matched normal 110/111 53 45 113/112 None None 21 Black CIN 1/matched normal 132/134 53 Variant Candidate 89 * 36 Black CIN 1/matched normal 147/149 52 None * 17 Black CIN 1/matched normal 157/159 18, 52, 56 52, 56 158/160 18, 52, 56 52, 56 22 White CIN 1/matched normal 176/177 53 53, 16 * 25 Black CIN 2/matched normal 130/136 31 None 131/137 None None 21 Black CIN 2/matched normal 151/153 16 Variant 16 * 24 White CIN 2/matched normal 221/219 52 52 222/220 52 52 46 White CIN 2/matched normal 235/233 None 18 234/236 18 58 21 White CIN 3/matched normal 114/116 16 16 * 28 White CIN 3/matched normal 141/143 16 59 142/144 None None 66 White CIN 3/matched normal 161/163 31 31 162/164 None None 30 White CIN 3/matched normal 165/166 16 16 * 39 Black CIN 3/matched normal 122/123 18, 58 58 * c 45 Black CIN 3/matched normal 216/218 16 18 * 19 White CIN 3/matched normal 241/243 16 None * 50 White SCCA/matched normal 181/182 45 45 183/185 None None 56 White SCCA/matched normal 193/195 31 31, 18 * 54 White SCCA/matched normal 197/199 16, 45 45 198/200 45 45 73 White SCCA/matched normal 201/203 16 16 * 35 White SCCA/matched normal 207/208 16 16 * 37 White SCCA/matched normal 237/239 18 Variant None *

* RNA samples not used for microarray. cAll lines represent a different individual except specimens 214, 216, and 218 that are from the same patient.

normal tissue as a control for each abnormal specimen. Using a The results of these experiments also suggest that a potential linear mixed model for analysis of gene expression patterns, we communication exist between stromal fibroblasts and premalig- propose the first genomically based model of early events in nant epithelial cells to induce proangiogenic signals that appear at cervical neoplastic transformation (Fig. 3). This application of a the CIN 2 transition. This analysis suggests that the ‘‘angiogenic linear mixed model is ideal for the complicated experimental switch,’’ as proposed by Hanahan and Folkman, may depend on design structure to control for interpatient gene expression profile cross-talk between stromal fibroblasts and CIN cells in the context variation. This ANOVA-based statistical analysis not only allows for of the differing tumor microenvironments (11). These authors accommodating for important covariants, such as age and race, suggest that the angiogenic switch may be critically important and but also estimates and separates the large between-patient a potentially rate-limiting step initiating early tumor progression as variation fromthe total variation and thus increases the statistical well as tumor establishment. These results extend this idea and power. Bioinformatic analysis of our results identified distinct suggest that the angiogenic switch in CIN 2 requires communica- expression pathways that coincided with the histopathologic tion between both epithelial cells and the surrounding stromal transitions fromnormalto CIN 1, CIN 2, CIN 3, and cancer. Based fibroblasts and that each compartment individually induces the on these results, we propose an initial evolving in vivo model for expression of angiogenic factors. cervical carcinogenesis (Fig. 3) that we hope will serve as a temp- The proproliferative genomic signature for the CIN 1 late for other researchers to build more sophisticated models as transition. Early infection of the cervix with HPV should induce additional and updated bioinformatics data become available (raw a series of molecular events in which the viral genome establishes data for this analysis can be obtained online). itself as an episome within basal epithelial cells and uses host www.aacrjournals.org 7119 Cancer Res 2007; 67: (15). August 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Cancer Research replication machinery to increase its copy number. The natural proliferation and ultimately transformation. Thus, we propose viral life cycle requires the expression of the viral oncogenes E6 and that the up-regulation of CDKN2A in early CIN and cervical E7, which dysregulate the host cell cycle by interacting with p53 cancer might be the host’s response to inactivation of pRb and and pRb, respectively. The histopathologic presentation that release of E2F. corresponds to early viral infection is referred to as the VCE It is also proposed that the HPV life cycle should activate (koilocytotic atypia) and CIN 1. Our data suggest an initial series of machinery that induces processes for viral DNA transport, genetic events in the basal epithelial cells that directly allows for packaging, and subsequent viral shedding. In this regard, KIF23 viral replication that may also indirectly establish an environment and CENPF are increased the CIN 1 transition. KIF23 is a permissive for neoplastic progression. Nine genes were validated by microtubule-dependent molecular motor that is essential for PCR (Table 3) and fall into two function physiologic groups: (a) midzone/midbody formation at the mitotic spindle during genes that favor cellular proliferation (CDKN2A, CENPF, KIF23, cytokinesis (13). Interestingly, KIF23 is also increased in PAP ITGAV, and ACAA1); and (b) genes that alter the cellular immune smears women with SCCA (Supplementary Fig. S2). CENPF is a system( IFNAR1, IL1RN, EMP1, and L1RN). These genes were used centromere passenger protein that acts as a scaffold to recruit and to propose a proproliferative/immunosuppression genomic signa- assemble kinetochores during the cell cycle. It ensures proper ture that corresponds with the CIN 1 transition (Fig. 3). segregation of chromosomes by facilitating interactions between Viral replication requires cell entry into the S-phase of cell cycle the centromere and mitotic spindle and overexpression of CENPF and expression of the E7 protein inactivates pRb resulting in the has been associated with human cancers (14). Thus, we suggest release of ‘‘unchecked’’ E2F family transcription factors that favor that the induction KIF23 and CENPF are necessary for the early progression through the late G1 cell cycle checkpoint (11). An viral life cycle and this may indirectly establishes a transformation- intercellular redundancy (or secondary) pathway that also inhibits permissive microenvironment. pRb activity involves phosphorylation by the CDK4/cyclinD1 Our microarray analysis showed that the integrin ITGAV complex and this process is negatively regulated by CDKN2A increases during early HPV infection and sharply increases at the (p16INK4a), whose primary action is to inhibit CDK4.Up- CIN 3 interphase. Integrins are heterodimeric membrane proteins regulation of CDKN2A in early CIN and cervical cancer might that regulate cell adhesion, cancer invasion, and altered patterns of represent the host’s response to inactivation of pRb (12). This is integrin expression have been reported in neoplastic epithelium. consistent with the idea that infected cells may inhibit both For example, integrin aVh3 has been implicated in the inhibition primary and secondary pathways that normally prevent cell of anoikis (15); thus, up-regulation of ITGAV might contribute to division and thus create a permissive state that is favorable for the survival of HPV-infected cells that rise through the suprabasal

Table 3. Up- and down-regulated genes alter in the CIN 1-3 transition signatures

CIN signature

CIN 1 CIN 2 CIN 3

Proproliferative/immunosuppression signature Proangiogenic stromal/epithelial interaction signature Proinvasive genomic signature

Proproliferative genes Proangiogenic genes Proinvasive genes

CDKN2A (p16INK4a)* Stromal genes DSG3 (desmoglein 3)* KIF23* HINT1* MMP3* CENPF* TAGLN2* MMP14* c TBX19 BRWD1* c DAB2 TNFRSF12A (TWEAK R)* c MAP2K7

Immunosuppression genes Epithelial genes Other genes

c IFNAR1 (IL receptor 1) KAL1* NOMO2* IL1RN (IL receptor antagonist)* TLOC1* c EMP1 c IL1RN

Other genes

ITGAV* c ACAA1

* Up-regulated. cDown-regulated.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Multidimensional Model of Cervical Carcinogenesis epithelium. Indeed, HPV-infected cells have been found to enter the immune system that may also indirectly establish permissive S-phase after they detach from the basal membrane (16), whereas microenvironment for transformation (Fig. 3). These genes were uninfected basal cells exit the cell cycle after losing contact with used to propose an immunosuppression genomic signature for the the basement membrane (Fig. 3). Thus, altered integrin expression CIN 1 transition. may favor HPV survival (Fig. 3). The analysis of these results suggests that the epithelial The expression of acetyl-CoA acyltransferase 1 (ACAA1) was also genomic effects of early HPV viral infection, in the transition to increased in CIN 1 epithelial cells. ACAA1 is operative in the h- koilocytotic atypia and CIN 1, share a very similar profile and oxidation systemof the peroxisomesand has never been these genes fall into two major categories. The first involves the implicated in tumorigenesis. However, peroxisome proliferator- uncoupling of cell cycle regulation that promotes proliferation activated receptors (PPAR) act as transcription factors, binding (CDKN2A, KIF23, CENPF, ITGAV, and EMP1) and the second with retinoid X receptor (RXR), and have been implicated in appears to allow virally infected cells to evade the host immune tumorigenesis (17). Moreover, PPARg has been shown to be down- system( IFNAR1 and IL1RN). This seems logical because after viral regulated in cervical tumors (18). Because ACAA1 is regulated by infection of a host cell, it should both initiate intracellular PPAR, we speculate that a common PPAR ligand, coactivator, or processes necessary for viral replication and at the same time RXR regulatory mechanism may be involved in the progression of hide itself from the host immune surveillance systems. Thus, we early cervical neoplasia (Fig. 3). Finally, these results suggest that refer to this transition as the ‘‘proproliferative/immunosuppres- these five genes (CDKN2A, CENPF, KIF23, ITGAV, and ACAA1)may sion genomic signature.’’ play a role in the natural viral life cycle by supporting S-phase entry, The proangiogenic stroma/epithelium interaction genomic proliferation, DNA segregation, and HPV survival and based on signature at the CIN 2 transition. This study suggests that during these results we propose a proproliferative early CIN 1 genomic the CIN 2 transition, epithelial cells may be responding to the lack signature. of local nutrients via a communication between stromal fibroblasts The immunosuppression genomic signature for the CIN 1 and epithelial cells to activate neovascularization pathways. This transition. It has been previously suggested that the early HPV life may result from the increasing metabolic demands from rapidly cycle should create a microenvironment that allows infected cells dividing epithelial cells that begin to overwhelmthe existing to conceal their presence from the host’s immune system. This vascular network. At the CIN 2 transition, six genes were validated analysis identified four genes (Table 3) that have been associated by PCR (Table 3) that all fall into a physiologic group that may with the regulation of the immune system (IFNAR1, IL1RN, EMP1, affect angiogenesis. The expression of five of these genes (Table 3) and L1RN). IFNAR1 encodes a membrane protein that stimulates was altered in stromal cells (HINT1 and TAGLN2 were up-regulated, Janus-activated kinase/signal transducers and activators of tran- whereas MAP2K7, DAB2, and TBX19 were down-regulated), whereas scription (JAK/Stat) signaling when complexed by IFNAR2 and only one gene was altered in epithelial cells (KAL1 was increased). bound by IFN-a (19). The IFN-a–JAK/Stat signaling pathway is HINT1 is a member of the conserved histidine triad protein critical for host defense against viral infection by stimulating family involved in a variety of regulatory networks including transcription of antiviral, antiproliferative, and antitumor genes. chromatin remodeling, proliferation, and immune response (28). This suggests a mechanism whereby increased IFNAR1 is up- HINT1 is a repressor of MITF, an essential mast cell protein that regulated in HPV-infected cells as a host feedback response to regulates expression of many important mast cell products, inhibition of the IFN-a-JAK/Stat pathway, as happens in a multiple including the mast cell proteases mMCP-5 and mMCP-6. In the myeloma cell line (20). This result is consistent with the induction murine model of cervical cancer, angiogenesis was activated of IFN-inducible genes identified in infected cell lines and may be a concurrently with infiltration of the stroma by mast cells and host response to infection (21). subsequent expression of mMCP-4 and mMCP-6 (29) and this is EMP1 expression is associated with squamous cell differentia- consistent with our results fromcervical biopsies. Thus, we tion and is suppressed by retinoic acid receptor signaling (22), and propose that increased expression of HINT1 in CIN 2 stroma transfection studies have shown an S-phase arrest along with may be a marker of mast cell infiltration and inflammation that decreased cell growth (23). IL1RN is a member of the interleukin 1 promotes vascularization and angiogenesis (Fig. 3). (IL-1) cytokine family and functions as a competitive receptor We also detected up-regulation of TAGLN2, a homologue of antagonist blocking binding of IL-1. EMP1 and IL1RN may play a transgelin that is a marker for hepatocellular carcinoma (29). The role in cervical cells via interaction with the ligand gated ion function of TAGLN2 is largely unknown but the gene becomes up- channel, P2RX7 (Fig. 2). It has been shown that the binding of regulated as prostatic stromal fibroblasts transform into myofibro- EMP1 and other EMP proteins to the COOH-terminal domain of blasts in response to transforming growth factor h (TGF-h; ref. 30) and P2RX7 activates channel formation and membrane blebbing (24). may be associated with early formation of a permissive environment Furthermore, activation of P2RX7 results in extracellular secretion for epithelial cell transformation. Thus, TAGLN2 could be one of of IL1RN (25). The microarray analysis suggests that the concurrent several factors that influence angiogenesis and downstreamcell down-regulation of EMP1 and IL1RN may be affecting this invasion by contributing to a‘‘reactive stroma’’microenvironment that pathway, with a final downstreameffect of decreased IL1RN has been described histopathologically as desmoplasia (31). secretion. Because IL1RN competes with IL-1 at the IL-1 receptor, We also report that the stress kinase MAP2K7 is down-regulated decreased expression of this antagonist may result in the increased in CIN stroma. MAP2K7 is widely expressed and mediates the cell downstreameffects of IL-1. This mightinclude the inhibition of responses to proinflammatory cytokines and environmental apoptosis in HPV-infected cells and the activation of cancer stresses. This protein is a negative regulator of mast cell À À promoting nuclear factor-nB and c-Jun/activator protein-1, which proliferation, as shown in mkk7( / ) mast cells that are hyper- are increased in cervical cancer (26, 27). Based on these results, we proliferative (32). Therefore, down-regulation of MAP2K7 at the suggest that the altered expression of IFNAR1, IL1RN, EMP1, and CIN 2 transition may be involved in release of negative regulation L1RN may be necessary for the viral concealment from the host’s of cell growth, proliferation, and angiogenesis. www.aacrjournals.org 7121 Cancer Res 2007; 67: (15). August 1, 2007

DAB2 is a widely expressed adaptor molecule involved in several desmosomes. There have been no reports of desmosomal junctions receptor-medicated signaling pathways, including cellular organi- in stromal components; however, the existence of simplified zation and macrophage adhesion, and TGF-h was decreased in this desmoglein-containing desmosomes between adjacent periodontal analysis. The function of DAB2 in the stroma is less understood, ligament fibroblasts has been shown (37). Because this type of although it appears to be a negative regulator of integrin aIIb/h3 fibroblast is known to contract (for tooth eruption and repair of and thereby an inhibitor of integrin-mediated fibrinogen adhesion periodontal tissue), it was suggested that desmosomes protect gap in platelets and platelet activation (33). Thus, we suggest that DAB2 junctions against cell contraction or strong occlusal forces (38). In down-regulation in the stroma may be a proangiogenic mechanism light of this evidence, we hypothesize a role for such modified leading to increased platelet activation and as such may establish a desmosomes in the fibroblasts of reactive stroma that may microenvironment permissive to vascularization, invasion, and establish a microenvironment favorable for cell invasion. potentially metastasis. In this analysis, two metalloproteinases, MMP3 and MMP14, Decreased expression of TBX19, a transcriptional activator of were increased at the CIN 3 transition (Fig. 3). Metalloproteinases POMC, which encodes polypeptide hormone precursors, such as function by degrading specific components of the extracellular the a-melanocyte–stimulating hormone (a-MSH), was also ob- matrix and it would seem logical to assume that their increased served. a-MSH induces mast cell apoptosis (34) via signaling expression might be responding to tumor cell overcrowding. MMP3 transduced by its receptor, MC1R. Reduced stromal expression of increases fromCIN 1 in epitheliumand then very sharply increases TBX19 might decrease the production of a-MSH, thereby in CIN 3 and SCCA tumor cells and stroma. MMP3 is an promoting mast cell survival and production of inflammatory extracellularly secreted matrix metalloproteinase that is overex- and angiogenic cytokines that should induce neovascularization. pressed in a variety of tumor types (39) and has a wide range of One example of an epithelial gene that may be responding to specificity for both extracellular matrix and nonmatrix substrates, nutrient deprivation is KAL1 (Table 3), which encodes anosmin-1, a suggesting extensive effects on tumorigenesis (40). Because MMP3 secreted protein found in the basement membrane and interstitial contributes to angiogenesis by releasing sequestered angiogenic matrices of multiple tissues during organogenesis (35). Anosmin-1 growth factors, it may be responding to the stress of nutrient enhances the cellular response to FGF2 by acting as a modulatory deprivation as premalignant cells begin to overcrowd. Similar to coligand that binds the FGFRI-FGF2-HSPG complex (Fig. 3). MMP3, MMP14 is also a marker for invasion that has been shown Increased KAL1 expression in cervical epitheliumshould result in to confer invasive ability to both normal and neoplastic cells and protein secretion that locally mediates angiogenesis by directly recently detected in high-grade cervical dysplasia as well as interacting with underlying stromal cells. This idea is consistent with invasive cervical cancer (41). MMP14 activates pro-MMP2, which is several studies that have detected an increase in angiogenic growth overexpressed in premalignant and malignant lesions of the cervix factors and microvessel density in cervical cancer (21, 36). Thus, this and associates with increased invasive potential (42). result suggests a complex interplay between stromal fibroblasts and BRWD1, a proposed transcriptional regulator involved in chromatin epithelial cells that may provide diffusible signals to initiate remodeling via interaction with SMARCA4 (a component of the SWI/ proangiogenic cascades responding to a lack of adequate nutrients. SNF complex), is overexpressed in stroma at the CIN 3 transition. The results presented above suggest that the predominant SMARCA4 is required to recruit Sp1, AP2, and polymerase II to the cellular and physiologic stress at the CIN 2 transition may be MMP2 promoter and enhances transcription (43). These results sug- nutrient deprivation resulting fromthe increased metabolic gest that induction of BRWD1 in tumor stroma may contribute to the demands of cell division. This seems like a logical assumption invasive phenotype indirectly by increasing the expression of spe- because the histologic appearance of CIN 2 shows an increase in cific downstream matrix metalloproteinases such as MMP2 (Fig. 3). the overall number of dividing cells that are beginning to fill the We also observed up-regulation of the TWEAK receptor, TNFRSF12A entire space above the basement membrane. These results also (FN14) that is a member of the TNF superfamily. TNFRSF12A stimu- may suggest that a fibroblast/epithelial cell communication or lates proliferation, migration/invasion, and may also play a role in interaction may be critical in how CIN 2 cells respond to a shortage tumor angiogenesis (44, 45). Thus, we suggest that the induction of of nutrients by inducing angiogenic factors that may be a more metalloproteinases either directly or indirectly at the CIN 3 transition generalized intermediate step in carcinogenesis. Thus, we propose is in response to cellular over crowding. that the CIN 2 transition is driven by metabolic demands and thus, It is well established that abnormal CIN 3 cells histologically suggest a ‘‘proangiogenic stroma/epithelium genomic signature.’’ involve the full thickness of the epitheliumand this observation The proinvasive genomic signature for the CIN 3/SCCA along with the results discussed above suggest that the predom- transition. It seems logical to assume that, as cervical cells inant microenvironmental stress may be cellular overcrowding. progress fromCIN 2 (proangiogenic) to CIN 3 and SCCA, pressures Thus, it seems logical that these CIN 3 cells should induce factors from the local microenvironment should continue to the point to overcome this stress by removing any local physical barriers to where overcrowding might become the predominant cell stress. If expansion. In this regard, several differentially regulated genes were this is the case, then specific genes that function to modulate and identified that may induce an invasive phenotype by altering overcome this cellular stresses should be activated. In this analysis, barriers to tumor cell expansion either directly (MMP3 and seven genes were validated by PCR (Table 3) and five of these fall MMP14) or indirectly (DSG3, BRWD1, NOMO2, and TNFRSF12A; into a functional class of proteins involved in invasion (DSG3, Fig. 3). Thus, we propose that the transition into CIN 3 be refer to MMP3, MMP14, BRWD1, and TNFRSF12A). Thus, based on these as the ‘‘proinvasive genomic signature.’’ results, it seemed logical to hypothesize that the predominant cellular stress present at the CIN 3 transition may be due to cellular overcrowding (Fig. 3). Conclusions DSG3 is down-regulated in the epitheliumand is up-regulated in The genomic results in this study were developed based on the stroma at the CIN 3 transition and is a key component of changes in gene expression in a linear manner that were

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Multidimensional Model of Cervical Carcinogenesis correlated with histologic changes to formulate an in vivo model transformation where abnormal epithelial cells are experiencing for cervical carcinogenesis. This model identifies three apparent overcrowding and the obvious response would be to eliminate any critical transitions. The first step appears to be consistent with physical barriers to cell expansion. Although this work validates the normal HPV life cycle after infection, including dysregulation many preexisting ideas for transformation in vitro, the real novelty of cell cycle regulation, which would like allow viral replication, is identification of the order of these events and their association and altering the host immune surveillance systems, so as the with specific CIN transitions that allows the construction of the virally infected cells might evade detection. The CIN 2 transition first in vivo model for cervical cancer. suggests a stromal/epithelial cell interaction that induces several signals to induce neovacuolization pathways. This would suggest an intermediate step in progression in which the accumulation of Acknowledgments CIN 2 cells, in an enclosed microenvironment, is outstripping the Received 1/19/2007; revised 2/28/2007; accepted 4/19/2007. Grant support: Intramural Research Program of the NIH, National Cancer local nutrient sources and the logical response would be to Institute, Center for Cancer Research, the Radiation Oncology Branch, and grants activate angiogenesis. DK51612 and CA82722; NIH grants CA95713 and CA94141; and Howard Hughes Finally, the transition into CIN 3 coincides with the induction of Medical Institute, where M.C. Funk is a medical student research fellow. The costs of publication of this article were defrayed in part by the payment of page factors that can disrupt physical barriers to tumor cell expansion charges. This article must therefore be hereby marked advertisement in accordance and invasion into neighboring tissues. This may be a late step in with 18 U.S.C. Section 1734 solely to indicate this fact.

References 16. Doorbar J, Foo C, Coleman N, et al. Characterization Similarities between tumor stroma generation and of events during the late stages of HPV16 infection wound healing. N Engl J Med 1986;315:1650–9. 1. Wright TC, Kurman RJ, Ferenczy A. Precancerous in vivo using high-affinity synthetic Fabs to E4. Virology 32. Sasaki T, Wada T, Kishimoto H, et al. The stress lesions of the cervix. In: Kurman RJ, editor. Blaustein’s 1997;238:40–52. kinase mitogen-activated protein kinase kinase (MKK)7 pathology of the female genital tract. New York: 17. Berger J, Moller DE. The mechanisms of action of Is a negative regulator of antigen receptor and growth Springer-Verlag; 1994. p. 229–77. PPARs. Annu Rev Med 2002;53:409–35. factor receptor-induced proliferation in hematopoietic 2. Walboomers JM, Jacobs MV, Manos MM, et al. Human 18. Jung T-I, Baek W-K, Suh S-I, et al. Down-regulation of cells. J Exp Med 2001;194:757–68. papillomavirus is a necessary cause of invasive cervical peroxisome proliferator-activated receptor g in human 33. Huang C-L, Cheng J-C, Liao C-H, et al. Disabled-2 is a h cancer worldwide. J Pathol 1999;189:12–9. cervical carcinoma. Gynecol Oncol 2005;97:365–73. negative regulator of integrin aIIb 3-mediated fibrino- 3. Sawaya GF, Brown AD, Washington AE, Garber AM. 19. Parmar S, Platanias L. Interferons: mechanisms of gen adhesion and cell signaling. J Biol Chem2004;279: Current approaches to cervical-cancer screening. N Engl action and clinical applications. Curr Opin Oncol 2003; 42279–89. J Med 2001;344:1603–7. 15:431–9. 34. Sarkar A, Sreenivasan Y, Manna SK. a-Melanocyte- 4. Baseman JG, Koutsky LA. The epidemiology of human 20. Walters D, Jelinek D. A role for Janus kinases in stimulating hormone induces cell death in mast cells: papillomavirus infections. J Clin Virol 2005;32:16–24. crosstalk between ErbB3 and the interferon-a signal- involvement of NF-nB. FEBS Lett 2003;549:87–93. 5. Castle PE, Schiffman M, Herrero R, et al. A prospective ing complex in myeloma cells. Oncogene 2004;23: 35. Hardelin J-P, Julliard AK, Moniot B, et al. Anosmin-1 study of age trends in cervical human papillomavirus 1197–205. is a regionally restricted component of basement acquisition and persistence in Guanacaste, Costa Rica. 21. Chang YE, Laimins LA. Microarray analysis identifies membranes and interstitial matrices during organogen- J Infect Dis 2005;191:1808–16. interferon-inducible genes and stat-1 as major tran- esis: Implications for the developmental anomalies of 6. Khan MJ, Partridge EE, Wang SS, Schiffman M. scriptional targets of human papillomavirus type 31. X chromosome-linked Kallmann syndrome. Dev Dyn Socioeconomic status and the risk of cervical intra- J Virol 2000;74:4172–82. 1999;215:26–44. epithelial neoplasia grade 3 among oncogenic human 22. Marvin KW, Fujimoto W, Jetten AM. Identification 36. Coussens LM, Raymond WW, Bergers G, et al. papillomavirus DNA-positive women with equivocal or and characterization of a novel squamous cell- Inflammatory mast cells up-regulate angiogenesis dur- mildly abnormal cytology. Cancer 2005;104:61–70. associated gene related to PMP22. J Biol Chem1995; ing squamous epithelial carcinogenesis. Genes Dev 1999; 7. Eisen MB, Brown PO. DNA arrays for analysis of gene 270:28910–6. 13:1382–97. expression. Methods Enzymol 1999;303:179–205. 23. Wang HT, Kong JP, Ding F, et al. Analysis of gene 37. Shore R, Berkovitz B, MoxhamB. Intercellular 8. Chuang YY, Chen Y, Gadisetti, et al. Gene expression expression profile induced by EMP-1 in esophageal contacts between fibroblasts in the periodontal connec- after treatment with hydrogen peroxide, menadione, or cancer cells using cDNA microarray. World J Gastro- tive tissues of the rat. J Anat 1981;133:67–76. t-butyl hydroperoxide in breast cancer cells. Cancer Res enterol 2003;9:392–8. 38. Yamaoka Y, Sawa Y, Ebata N, Yoshida S, Kawasaki T. 2002;62:6246–54. 24. Wilson HL, Wilson SA, Surprenant A, North RA. Desmosomal proteins in cultured and intact human periodontal ligament fibroblasts. Tissue Cell 1999;31:605–9. 9. Benjamini Y, Hochberg Y. Controlling the false Epithelial membrane proteins induce membrane bleb- 39. Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, discovery rate: a practical and powerful approach to bing and interact with the P2X7 receptor C terminus. Bissell MJ. Matrix tetalloproteinase stromelysin-1 trig- multiple testing. J R Stat Soc Ser B 1995;57:289–300. J Biol Chem2002;277:34017–23. gers a cascade of molecular alterations that leads to 10. Li J, Gerhard DS, Zhang Z, et al. Denaturing high- 25. Wilson HL, Francis SE, Dower SK, Crossman DC. stable epithelial-to-mesenchymal conversion and a performance liquid chromatography for detecting and Secretion of intracellular IL-1 receptor antagonist (type 1) premalignant phenotype in mammary epithelial cells. typing genital human papillomavirus. J Clin Microbiol is dependent on P2X7 receptor activation. J Immunol J Cell Biol 1997;139:1861–72. 2003;41:5563–71. 2004;173:1202–8. 40. Egeblad M, Werb Z. New functions for the matrix 11. Hanahan D, Folkman J. Patterns and emerging 26. Nair A, Venkatraman M, Maliekal TT, Nair B, metalloproteinases in cancer progression. Nat Rev mechanisms of the angiogenic switch during tumori- Karunagaran D. NF-nB is constitutively activated in Cancer 2002;2:161–74. genesis. Cell 1996;86:353–64. high-grade squamous intraepithelial lesions and squa- 41. Zhai Y, Hotary KB, Nan B, et al. Expression of 12. Dray M, Russell P, Dalrymple C, et al. p16(INK4a) as a mous cell carcinomas of the human uterine cervix. membrane type 1 matrix metalloproteinase Is associat- complementary marker of high-grade intraepithelial Oncogene 2003;22:50–8. ed with cervical carcinoma progression and invasion. lesions of the uterine cervix. I. Experience with 27. Prusty BK, Das BC. Constitutive activation of Cancer Res 2005;65:6543–50. squamous lesions in 189 consecutive cervical biopsies. transcription factor AP-1 in cervical cancer and 42. Brummer O, Bohmer G, Hollwitz B, Flemming P, Petry Pathology 2005;37:112–24. suppression of human papillomavirus (HPV) transcrip- K-U, Kuhnle H. MMP-1 and MMP-2 in the cervix uteri in 13. Zhu C, Bossy-Wetzel E, Jiang W. Recruitment of tion and AP-1 activity in HeLa cells by curcumin. Int J different steps of malignant transformation—an immu- MKLP1 to the spindle midzone/midbody by INCENP is Cancer 2005;113:951–60. nohistochemical study. Gynecol Oncol 2002;84:222–7. essential for midbody formation and completion of 28. Weiske J, Huber O. The histidine triad protein Hint1 43. Ma X-J, Salunga R, Tuggle JT, et al. Gene expression cytokinesis in human cells. Biochem J 2005;389:373–81. triggers apoptosis independent of its enzymatic activity. profiles of human breast cancer progression. Proc Natl 14. de la Guardia C, Casiano CA, Trinidad-Pinedo J, Ba´ez J Biol Chem2006;281:27356–66. Acad Sci U S A 2003;100:5974–9. A. Cenp-F gene amplification and overexpression in 29. Shi Y, Wang H, Yin Y, et al. Identification and analysis 44. Winkles JA, Tran NL, Berens ME. TWEAK and Fn14: head and neck squamous cell carcinomas. Head Neck of tumour-associated antigens in hepatocellular carci- New molecular targets for cancer therapy? Cancer Lett 2001;23:104–12. noma. Br J Cancer 2005;92:929–34. 2006;235:11–7. 15. Zhang Y, Lu H, Dazin P, Kapila Y. Squamous cell 30. Untergasser G, Gander R, Lilg C, Lepperdinger G, Plas 45. Tran NL, McDonough WS, Savitch BA, et al. Increased carcinoma cell aggregates escape suspension-induced, E, Berger P. Profiling molecular targets of TGF-B1 in fibroblast growth factor-inducible 14 expression levels p53-mediated anoikis: fibronectin and integrin av prostate fibroblast-to-myofibroblast transdifferentiation. promote glioma cell invasion via Rac1 and nuclear factor- mediate survival signals through focal adhesion kinase. Mech Ageing Dev 2005;126:59–69. {n}B and correlate with poor patient outcome. Cancer Res J Biol Chem2004;279:48342–9. 31. Dvorak H. Tumors: Wounds that do not heal. 2006;66:9535–42. www.aacrjournals.org 7123 Cancer Res 2007; 67: (15). August 1, 2007

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Profiling Microdissected Epithelium and Stroma to Model Genomic Signatures for Cervical Carcinogenesis Accommodating for Covariates

David Gius, Margo C. Funk, Eric Y. Chuang, et al.

Cancer Res 2007;67:7113-7123.

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