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Cancer Genes and Genomics

Frequent HIN-1 Promoter Methylation and Lack of Expression in Multiple Human Tumor Types¹

¹ Department of Medical Oncology, Dana-Farber Cancer Institute, ² Harvard Medical School, and ³ Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts; ⁴ Laboratory of Population Genetics, National Cancer Institute, Bethesda, Maryland; ⁵ University of Nebraska Medical Center, Omaha, Nebraska; ⁶ Institute of Cancer Genetics, Department of Pathology, Columbia University, New York, New York; ⁷ Stanford University, Stanford, California; and ⁸ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Requests for reprints: Kornelia Polyak, Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, D740C, Boston, MA 02115. Phone: 617-632-2106; Fax: 617-632-4005. E-mail: Kornelia_Polyak{at}dfci.harvard.edu

	Abstract

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
HIN-1 (high in normal-1) is a candidate tumor suppressor identified as a gene silenced by methylation in the majority of breast carcinomas. HIN-1 is highly expressed in the mammary gland, trachea, lung, prostate, pancreas, and salivary gland, and in the lung, its expression is primarily restricted to bronchial epithelial cells. In this report, we show that, correlating with the secretory nature of HIN-1, high levels of HIN-1 protein are detected in bronchial lavage, saliva, plasma, and serum. To determine if, similar to breast carcinomas, HIN-1 is also silenced in tumors originating from other organs with high HIN-1 expression, we analyzed its expression and promoter methylation status in lung, prostate, and pancreatic carcinomas. Nearly all prostate and a significant fraction of lung and pancreatic carcinomas showed HIN-1 hypermethylation, and the majority of lung and prostate tumors lacked HIN-1 expression. In lung carcinomas, the degree of HIN-1 methylation differed among tumor subtypes (P = 0.02), with the highest level of HIN-1 methylation observed in squamous cell carcinomas and the lowest in small cell lung cancer. In lung adenocarcinomas, the expression of HIN-1 correlated with cellular differentiation status. Hypermethylation of the HIN-1 promoter was also frequently observed in normal tissue adjacent to tumors but not in normal tissue from noncancer patients, implying that HIN-1 promoter methylation may be a marker of premalignant changes. Thus, silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that elimination of HIN-1 function may contribute to several forms of epithelial tumorigenesis.

	Introduction

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
HIN-1 (high in normal-1) was identified by serial analysis of gene expression as a gene highly expressed in normal luminal mammary epithelial cells and down-regulated in in situ, invasive, and metastatic breast carcinomas (1). The silencing of HIN-1 expression in the majority of breast tumors was found to be due to methylation of the proximal promoter and first exon of the HIN-1 gene (1). Similar results were reported recently for primary nasopharyngeal carcinomas (2). The high frequency of loss of HIN-1 expression in human breast carcinomas suggested a tumor suppressor function, and correlating with this, reintroduction of HIN-1 into breast cancer cells inhibited cell growth (1). During mouse embryonic development, the expression of HIN-1 was associated with the terminal differentiation of tracheobronchial epithelial cells (3). In addition, HIN-1 was up-regulated by retinoic acid–induced differentiation of human bronchial epithelial cells, suggesting a role for HIN-1 in mucinous epithelial cell differentiation (3). Correlating with this, a homologue of HIN-1, uteroglobin-related protein-1 (UGRP-1), was identified in the mouse as a target of the Nkx2.1 homeogene that is required for lung development and differentiation (4, 5). UGRP-1 was found to have a limited homology to uteroglobin; thus, both HIN-1 and UGRP-1 are considered to be distant members of the secretoglobin family and are designated as secretoglobin 3A1 (SCGB3A1) and secretoglobin 3A2 (SCGB3A2), respectively.

To further dissect the role of HIN-1 in epithelial cell function and tumorigenesis, we analyzed its expression in various normal human organs and body fluids. In addition, we determined HIN-1 expression and promoter methylation status in lung, prostate, and pancreatic tumors.

	Results and Discussion

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
HIN-1 in Normal Lung Tissue and Body Fluids
We have determined previously that human HIN-1 is highly expressed in the adult mammary gland, lung, trachea, pancreas, prostate, and salivary gland (1). In mouse, the highest HIN-1 expression is detected in the lung, with much lower levels observed in other organs including the mammary gland (3, 4, 6). In the mouse lung, the HIN-1 message is localized to the epithelial cells of the trachea, bronchi, and bronchioli, and during embryonic development, HIN-1 mRNA levels correlate with the terminal differentiation of these cells (3). To determine the expression of HIN-1 in human lung tissue at the cellular level, we did mRNA in situ hybridization analysis. As depicted in Fig. 1A, similar to mouse, the HIN-1 message is specifically localized to bronchial epithelial cells, whereas lower levels are detected in some pneumocytes in the lung parenchyma. No HIN-1 expression was detected in type I epithelial cells of the distal alveolar sacs consistent with proximal mucociliary and distal alveolar epithelial cells being derived from a different lineage during development (7), although some reactive type II epithelial cells showed sparse HIN-1 expression (Fig. 1A). In addition, not all proximal bronchial epithelial cells expressed HIN-1, but it is currently unknown if HIN-1-expressing cells represent a specific cellular subtype.

View larger version (67K): [in this window] [in a new window]   FIGURE 1. HIN-1 expression and protein levels in human lung and body fluids. A. mRNA in situ hybridization with antisense probe (red) showing high and specific HIN-1 expression in a subset of bronchial epithelial cells and sparsely scattered reactive type II cells in the lung parenchyma. No signal is detected with the sense probe. B. HIN-1 immunoblot analysis of preimmune (P) or HIN-1 (H) immunoprecipitates of saliva, bronchial wash, plasma, and serum from several independent individuals. The endogenous HIN-1 protein migrates as a smear at 10 kDa and is highly abundant in all these body fluids. C. mRNA in situ hybridization of lung adenocarcinomas and squamous tumors showing high HIN-1 expression in normal bronchial epithelial cells in tumors 256 and 57 (arrows) and in epithelial cells of well-differentiated parts of the two adenocarcinomas. Adjacent moderately differentiated areas of the two adenocarcinomas and all squamous tumor cells lack HIN-1 expression. D. Reverse transcription-PCR analysis of HIN-1 expression in different parts of normal prostate (LPZ, left peripheral zone; RPZ, right peripheral zone; CZ, central zone), benign prostatic hyperplasia (BPH), and prostate carcinomas (T1–T7). High HIN-1 expression is detected in all areas of the normal prostate and in benign prostatic hyperplasia, whereas most prostate tumors lack HIN-1 expression. Amplification of the ACTB gene was used as control.

HIN-1 Expression in Lung and Prostate Tumors
To determine if, as observed in breast cancer, HIN-1 expression is lost or down-regulated in lung carcinomas, we did Northern blot analysis of 27 lung adenocarcinomas and 12 squamous lung tumors. Most of the tumors showed no or very low levels of HIN-1 expression compared with normal lung tissue (data not shown). Because these tumors were not microdissected, even the observed low level of HIN-1 expression could be due to contaminating normal bronchial epithelial cells. To evaluate the expression of HIN-1 in more detail, we used mRNA in situ hybridization to determine the expression of HIN-1 at the cellular level. All 8 squamous tumors and 8 of 10 adenocarcinomas analyzed completely lacked HIN-1 expression, whereas the well but not the moderately differentiated parts of the remaining 2 adenocarcinomas showed high HIN-1 levels (Fig. 1C and data not shown). These results correlate well with our previous results demonstrating a link between HIN-1 expression and mucinous differentiation of bronchial epithelial cells (3). Correlating with our results, a recent study reported that HIN-1 expression is frequently down-regulated in stage I non-small lung carcinomas and that this correlates with poor clinical outcome (8).

The decreased HIN-1 expression in lung tumors could suggest that these tumors originate from a cell type that does not normally express HIN-1 or that its expression is silenced during tumorigenesis. Adenocarcinomas and squamous carcinomas are frequently localized to the distal and proximal lung, respectively; they have distinct gene expression profiles (9-11) and genetic changes (12); and they are presumed to arise from different cell types (9, 11, 13, 14).

To analyze HIN-1 expression in different parts of the normal prostate and in prostate tumors, we did reverse transcription-PCR analysis of the left and right peripheral and central zones of normal prostate, a benign prostatic hyperplasia, and seven prostate carcinomas. A high level of HIN-1 expression was detected in all zones of normal prostate and in benign prostatic hyperplasia, whereas most prostate carcinomas lacked HIN-1 expression (Fig. 1D).

Methylation of HIN-1 in Normal and Cancerous Lung, Prostate, and Pancreatic Tissues and Cell Lines
In breast carcinomas, we found a strong correlation between lack of HIN-1 expression and hypermethylation of its promoter region, suggesting that methylation is responsible for silencing HIN-1 expression in tumors (1). Recently, frequent HIN-1 promoter methylation and associated loss of expression were also reported in nasopharyngeal carcinomas (2). To determine if HIN-1 promoter methylation also occurs in other cancer types that lack HIN-1 expression, we first analyzed the sequence of the HIN-1 proximal promoter and first exon in cancer cell lines and the normal and cancerous tissues from various organs. This analysis showed that the HIN-1 promoter region is heavily methylated in two prostate cancer cell lines, whereas more moderate levels of methylation were seen in primary lung, prostate, and pancreatic carcinomas and a pancreatic cancer cell line (Fig. 2A). None of the prostate and pancreatic cancer cell lines showed significant levels of HIN-1 mRNA, suggesting that loss of expression is associated with promoter methylation.

View larger version (61K): [in this window] [in a new window]   FIGURE 2. Analysis of methylation patterns of the HIN-1 proximal promoter region and first exon in various cell and tissue types. A. Results of sequence analysis of bisulfite-treated genomic DNA from the indicated cell and tissue types. ZR75-1-AC, 5azaC-treated cells; BEC, primary bronchial epithelial cells. Circles, potential methylation sites (CpG); black and white, frequency at which the site was found to be methylated in the clones analyzed (, 0%; , 25%; , 50%; , 75%; , 100%). + and -, HIN-1 mRNA levels; +++, high level of expression detected in normal mammary epithelial cells; +, mRNA levels detectable by Northern blot analysis of total RNA (5 µg). Location of the previously published (arrows below circles) and new (arrows above circles; open arrow, forward primer use for detection of unmethylated DNA) sets of primers as well as the results of MSP. B. MSP analysis of the HIN-1 promoter region in lung tumors (T) and corresponding adjacent nonmalignant (N) lung tissue. Lower panel, results in tumors that were used for mRNA in situ hybridization (Fig. 1C). M and U, amplification using methylated and unmethylated sequence-specific primers, respectively. C. MSP analysis of the HIN-1 promoter region in normal lung, prostate, and pancreas obtained from noncancer patients (N1–N7). M and U, amplification using methylated and unmethylated sequence-specific primers, respectively.

Based on the methylation and gene expression information obtained from the HIN-1 promoter sequence and reverse transcription-PCR analyses, respectively, the frequency of methylated CGs seemed to be higher at more distal (–120 to –300 bp) parts of the promoter in lung, prostate, and particularly in pancreas compared with breast carcinomas, and this correlated most consistently with lack of HIN-1 expression (Fig. 2A). Therefore, we designed a different set of primers and developed a more distal HIN-1 promoter-specific methylation-specific PCR (MSP) assay (15) instead of using the one we have used previously for breast tumors (1). The position of the new and previously used MSP primers is depicted in Fig. 2A. We compared the newly designed MSP primers with the previously used and published set on a series (94 samples) of breast carcinomas and found that they gave nearly identical results (concordance 95%). In addition, when we compared them side by side using genomic DNA prepared from prostate cancer cell lines (PC3 and LNCaP) and primary lung, prostate, and pancreatic carcinomas, we found that, although the results were concordant, the new primers worked more consistently on samples with a limited amount of DNA and produced evaluable and reproducible results in a much higher fraction of samples. Based on these observations, we chose to use the new primers in all subsequent experiments.

Next, we analyzed the methylation status of HIN-1 in primary lung, prostate, and pancreatic carcinomas, corresponding normal tissues, and cancer cell lines using MSP. Similar to breast carcinomas, a significant fraction of lung, prostate, and pancreatic carcinomas showed complete or partial methylation (Fig. 2; Table 1). The highest frequency of HIN-1 methylation was observed in prostate cancer, wherein all xenografts (9 of 9) and almost all (20 of 21) primary tumors were methylated. Similarly, all pancreatic cancer cell lines (10 of 10) and most primary tumors (9 of 17) showed HIN-1 methylation (Table 1). Most importantly, the HIN-1 promoter was highly methylated in all lung and prostate tumors that lacked HIN-1 expression based on in situ hybridization (Fig. 1C and data not shown) and reverse transcription-PCR (Fig. 1D), respectively. Thus, similar to breast and nasopharyngeal carcinomas, HIN-1 promoter methylation seems to correlate with its lack of expression in lung and prostate carcinomas.

In the lung, the overall frequency of HIN-1 methylation was somewhat lower and varied according to cancer type. The lowest HIN-1 methylation frequency was observed in small cell lung cancer and the highest in squamous tumors (Table 1). The differences in HIN-1 methylation frequencies among the three lung cancer subtypes were statistically significant (P = 0.02), suggesting that different lung tumors may originate from different cell types and/or have distinct tumorigenesis pathways. The finding that the highest level of HIN-1 methylation is found in squamous lung carcinomas is particularly interesting in light of the fact that these tumors are frequently proximal, as is HIN-1 expression in normal lung, and that in normal bronchial epithelial cells the expression of HIN-1 is down-regulated in cells that have lost their mucinous differentiation phenotype and acquired squamous features (3). Correlating with our HIN-1 methylation results, recent studies have described distinct clustering of lung and other carcinomas according to histologic subtypes based on the methylation profile of a set of genes known to be methylated in lung cancer and microarray-based DNA methylation analysis, respectively (18, 19). Similar to prostate and pancreatic tumors, we found that a high fraction of normal lung tissue adjacent to tumor was also methylated but, in all these cases, the matched tumor showed even higher levels of methylation and samples from noncancer patients were almost all unmethylated (Fig. 2B and C). Again, this suggests that the adjacent nonmalignant tissue may have had some contaminating cancer cells or that premalignant changes associated with HIN-1 methylation may have occurred in these adjacent tissues.

The observation that, in lung adenocarcinomas, the expression of HIN-1 correlated with the differentiation status of the cells (Fig. 1C) raised the question of whether down-regulation and methylation of HIN-1 simply reflects lack of differentiation or it is specifically associated with tumorigenesis. To distinguish between these possibilities, we analyzed the methylation status of the HIN-1 promoter in normal bronchial epithelial cells grown in the absence of retinoic acid resulting in loss of mucinous differentiation, acquisition of squamous characteristics, and lack of HIN-1 expression. No methylation was detected in these cells by MSP and sequencing analysis, suggesting that lack of the normal mucinous differentiation program in these cells by itself is not sufficient to lead to HIN-1 methylation (Fig. 2A and data not shown).

In summary, these results show that silencing of HIN-1 expression due to methylation occurs in multiple human cancer types originating from organs that normally have high HIN-1 expression levels. In lung carcinomas, HIN-1 methylation seems to be specifically associated with tumorigenesis and distinct histologic subtypes, potentially suggesting different cell type of origin or pathways of tumorigenesis for squamous carcinoma, adenocarcinoma, and small cell lung carcinoma.

	Materials and Methods

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
Cell Lines and Tissue Specimens
All human tissue specimens were collected at the Brigham and Women's Hospital, Johns Hopkins Medical Center, and NIH following NIH guidelines and using protocols approved by the institutional review boards. Human cancer cell lines were obtained from American Type Culture Collection (Manassas, VA) or generously provided by Dr. Steve Ethier (University of Michigan Medical Center, Ann Arbor, MI). Cells were grown in medium recommended by the provider.

mRNA In situ Hybridization and Northern Blot Analysis
To generate templates for in vitro transcription reactions, the full-length human HIN-1 cDNA was PCR amplified and subcloned into pZero 1.0 (Invitrogen, Carlsbad, CA) and was used for the generation of sense and antisense digitonin-labeled riboprobes followed by mRNA in situ hybridizations essentially as described previously (20). The hybridized sections were observed with a Nikon microscope, images were obtained using an Axio camera, and photographs were organized using the AxioVision software. Hybridizations were considered successful if the sense probe gave no significant signal. Northern blot analysis was done as described previously (1).

Immunoprecipitation and Immunoblot Analysis
For immunoprecipitation analysis, human body fluids were supplemented with salt and reagents to 500 mmol/L NaCl, 10% glycerol, 20 mmol/L Tris (pH 8), and 0.5% Triton X-100 final concentration and incubated with preimmune serum or anti-HIN-1 antibody coupled to protein A-Sepharose beads for 2 hours at 4°C followed by repeated washes in the same buffer. Immunocomplexes were resolved on 12% SDS NuPAGE gels (Invitrogen) and immunoblotted with anti-HIN-1 antibody as described previously (1).

Methylation Assays and Statistical Analysis
Genomic DNA preparations and bisulfite treatment were done as described previously (1). PCR primers used were as follows: MSP, unmethylated, forward primer 5'-ATTGTAAAGTGAAGGTGTGGGTT-3' and reverse primer 5'-CCAACTTCCTACTACAACCAACA-3'; methylated, forward primer 5'-GTTTAGTTTTGAGGGGGGCGC-3' and reverse primer 5'-AACTTCCTACTACGACCGACG-3'. The PCR conditions were as follows: 94 x 3 minutes, 92 x 20 seconds, 63 x 30 seconds, 72 x 30 seconds (5 cycles), 92 x 20 seconds, 60 x 30 seconds, 72 x 30 seconds (35 cycles), and 72 x 5 minutes. To amplify the promoter area for sequencing, the PCR conditions were as follows: 94 x 3 minutes, 92 x 20 seconds, 55 x 30 seconds, 72 x 60 seconds (5 cycles), 92 x 20 seconds, 58 x 30 seconds, 72 x 60 seconds (30 cycles), and 72 x 5 minutes. PCR fragments were subcloned into pZero 1.0 and at least four individual clones per fragment were sequenced to determine methylation frequency.

Statistical significance was calculated using two-sided Fisher exact tests.

	Acknowledgements

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
We thank Dr. Rebecca Gelman for statistical analysis, Dr. Dale Porter for critical reading of the manuscript, and Dr. Angelo M. DeMarzo and Helen Fedor of the Prostate Specimen Repository at Johns Hopkins University School of Medicine for providing prostate tissue samples.

	Notes

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
¹ National Cancer Institute SPORE in Breast Cancer at Dana-Farber/Harvard Cancer Center grant CA89393, NIH RO1 grant CA94074-01A1, NIH National Research Service Award CA94787-01, Dunkin' Donuts "Rising Stars" Award, and V Foundation.

Received March 19, 2003; revised June 8, 2004; accepted August 2, 2004.

	References

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krop, I. Articles by Polyak, K. Search for Related Content PubMed PubMed Citation Articles by Krop, I. Articles by Polyak, K.