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[Cancer Research 65, 9659-9669, November 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

HIN-1, an Inhibitor of Cell Growth, Invasion, and AKT Activation

Ian Krop1,3, Michele Taylor Parker1,3, Noga Bloushtain-Qimron1, Dale Porter1,3, Rebecca Gelman2,3, Hidefumi Sasaki4, Matthew Maurer5, Mary Beth Terry6, Ramon Parsons5 and Kornelia Polyak1,3

Departments of 1 Medical Oncology and 2 Biostatistics, Dana-Farber Cancer Institute; 3 Harvard Medical School, Boston, Massachusetts; 4 Department of Surgery, Nagoya City University Medical School, Nagoya, Japan; and Departments of 5 Medicine and 6 Epidemiology, Columbia University, New York, New York

Requests for reprints: Kornelia Polyak, 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
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The HIN-1 gene encoding a small, secreted protein is silenced due to methylation in a substantial fraction of breast, prostate, lung, and pancreatic carcinomas, suggesting a potential tumor suppressor function. The receptor of HIN-1 is unknown, but ligand-binding studies indicate the presence of high-affinity cell surface HIN-1 binding on epithelial cells. Here, we report that HIN-1 is a potent inhibitor of anchorage-dependent and anchorage-independent cell growth, cell migration, and invasion. Expression of HIN-1 in synchronized cells inhibits cell cycle reentry and the phosphorylation of the retinoblastoma protein (Rb), whereas in exponentially growing cells, HIN-1 induces apoptosis without apparent cell cycle arrest and effect on Rb phosphorylation. Investigation of multiple signaling pathways revealed that mitogen-induced phosphorylation and activation of AKT are inhibited in HIN-1–expressing cells. In addition, expression of constitutively activate AKT abrogates HIN-1–mediated growth arrest. Taken together, these studies provide further evidence that HIN-1 possesses tumor suppressor functions, and that these activities may be mediated through the AKT signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To identify molecular alterations involved in the initiation and progression of breast carcinomas, we analyzed the global gene expression profiles of normal mammary epithelial cells and in situ, invasive, and metastatic breast carcinomas using serial analysis of gene expression (SAGE; ref. 1). Many of the genes we identified as highly expressed in normal mammary epithelium (HIN genes for high in normal) and lost in carcinomas encoded secreted proteins, cytokines, and chemokines, implicating abnormal paracrine and autocrine signaling in the initiation of breast tumorigenesis. One of these genes, HIN-1, seemed particularly interesting, and due to its intriguing expression pattern and putative novel function, we characterized it in further detail (2). In subsequent experiments, we determined that the loss of HIN-1 expression in breast carcinomas is due to promoter hypermethylation in the majority of breast tumors (>70%; ref. 2). Constitutive expression of HIN-1 in human breast cancer cells inhibits colony growth (2). In addition to normal luminal mammary epithelial cells, HIN-1 is abundantly expressed in the epithelial cells of normal lung, as well as prostate, salivary gland, and pancreas (2, 3). We and others recently reported that in addition to breast cancer, HIN-1 expression is down-regulated in the majority of lung, prostate, pancreatic, and nasopharyngeal cancers and similar to that observed in breast cancer, this down-regulation is associated with hypermethylation of the HIN-1 promoter (4–7). Importantly, in non–small cell lung cancer, down-regulation of HIN-1 was the most significant independent predictor of poor clinical outcome in stage I disease, suggesting that loss of HIN-1 expression is functionally relevant to tumorigenesis (5). Thus, silencing of HIN-1 expression by methylation is an early and frequent event in multiple human cancer types, and along with the in vitro data on growth inhibition, suggests that HIN-1 is a candidate tumor suppressor gene.

Recently, uteroglobin-related protein 1 (UGRP1), a homologue of HIN-1, was identified as the target of the Nkx2.1 homeodomain transcription factor (8). Based on their amino acid sequence and predicted structure, both HIN-1 and UGRP1 belong to the secretoglobin family (9). Therefore, HIN-1 and UGRP-1 are now also named as SCGB3A1 and SCGB3A2 for secretoglobin 3A1 and 3A2, respectively (10). The prototypical member of the secretoglobin superfamily is uteroglobin, a small secreted protein highly expressed in the epithelial cells of normal lung, uterus, breast, and prostate (11). Similar to HIN-1, the expression of uteroglobin is lost in the majority of lung and prostate cancers (11). Exogenous overexpression of uteroglobin in cancer cell lines results in reversion of many features of the transformed phenotype, including decreased anchorage-dependent and anchorage-independent growth as well as decreased ability to invade extracellular matrix (12–15). Furthermore, mice with homologous deletion of the uteroglobin gene develop spontaneous tumors and show increased susceptibility to chemical carcinogen–induced lung cancer (16, 17). Taken together, these data suggest that uteroglobin acts as a tumor suppressor gene and, based on the structural and functional similarities between uteroglobin and HIN-1, provide further rationale to explore potential tumor suppressor activities of HIN-1.

In this study, we further characterized the function and mechanism of action of HIN-1 in human immortalized mammary epithelial cells and breast cancer cell lines. We report that HIN-1 is a potent inhibitor of anchorage-dependent and anchorage-independent cell growth, migration, and invasion and induces cell cycle arrest and apoptosis. Data is presented suggesting that HIN-1 acts through a high-affinity cell surface receptor present on epithelial cells, and that the mechanism of action of HIN-1 involves modulation of the AKT signal transduction pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples and cell lines. Breast cancer, VA-13 fibroblast, and 293T cell lines were obtained from the American Type Culture Collection (Manassas, VA), HME50 normal immortalized mammary epithelial (18) and H157 and H1355 lung cancer cell lines were a generous gift of Dr. Jerry Shay (University of Texas) and Dr. Fred Kaye (National Cancer Institute), respectively. All cell lines were grown in medium recommended by the provider. Fresh, frozen, or formalin-fixed, paraffin-embedded tumor specimens were obtained from the Brigham and Women's Hospital (Boston, MA) and the National Disease Research Interchange (Philadelphia, PA). All human tissue was collected using protocols approved by the Dana-Farber Cancer Institute (DFCI) Institutional Review Board (Boston, MA). The TET-OFF inducible system was established in MDA-MB-435S cells by consecutive transfection of the pTA-IRES-NEO and pBI-EGPF-HIN-1 plasmids and selecting for positive clones. Two independent HIN-1–expressing clones were derived, one with enhanced green fluorescence protein (EGFP) expression (clone 7) and one without EGFP expression (clone 26) to control for potential effects due to EGFP overexpression. Neomycin- and myristoylated AKT (myrAKT)–expressing clones of MCF-7 and MDA-MB-435S and PDK1-overexpressing clones of MCF-7 and MCF-10A cells were established by infecting the cells with recombinant retroviruses.

Plasmid constructs and recombinant adenoviruses and retroviruses. Retroviral pWZL-myrAKT, pWZL-PI3K(p110), and pWZL-Neo plasmids were kindly provided by Drs. Jean Zhao and Tom Roberts (DFCI), whereas overexpression of PDK1 was achieved using pOZ-FH-N and transfected cells were isolated using anti-IL2R{alpha}-coupled magnetic beads. Retroviruses were generated and infections were carried out essentially as described previously (19). For ligand-binding assays, we generated a NH2-terminal alkaline phosphatase (AP) COOH-terminal HIN-1 fusion proteins using the AP-TAG-5 expression vector (GenHunter, Nashville, TN). Recombinant adenoviruses expressing HIN-1 were generated by subcloning human HIN-1 cDNA into the pShuttle-CMV transfer plasmid and using the Ad-Easy system for bacterial recombination (20). Large-scale generation and purification of adenoviruses was done as described (20). For the establishment of the TET-OFF inducible system, we used the pTA-IRES-NEO and pBI-HIN-1 constructs essentially as described (21). Plasmid constructs expressing wild-type and mutant p27 protein fused to GFP were kindly provided by Dr. Joyce Slingerland (Sunnybrook and Women's College Health Sciences Centre, Montreal, Canada).

RNA isolation and serial analysis of gene expression library generation and analysis. RNA was isolated from inducible MDA-MB-435 cells before and 24 and 48 hours after induction of HIN-1 expression and SAGE libraries were generated and analyzed essentially as previously described (2). SAGE data was deposited into the SAGE Genie database.7

Receptor and ligand binding assays. We did in vivo and in vitro ligand-binding assays on primary tissues and cell lines using AP-HIN-1 essentially as described (22). Briefly, frozen sections of various human specimens were fixed, incubated with either AP-HIN-1 fusion protein or AP control conditioned medium, washed, and then incubated with AP substrate forming a blue/purple precipitate. Coincubation of sections with 1,000x molar excess HIN-1 protein without AP fusion tag competed AP-HIN-1 binding (data not shown). For in vitro assays, we incubated cells in suspension with conditioned medium containing either AP alone or AP-HIN-1 fusion protein, washed cells, and then assayed for bound AP activity. Specificity of the binding was confirmed by using an excess of untagged HIN-1 or irrelevant secreted protein. Scatchard transformation was done as described (23). To determine if HIN-1 binds to lipophilic compounds, we generated a bacterial expression construct that expresses HIN-1 with a COOH-terminal tyrosine allowing iodination of purified recombinant HIN-1 protein (the human HIN-1 protein lacks tyrosine residues). Iodination was done essentially as described (23). Compounds were spotted onto a nitrocellulose membrane and incubated with iodinated recombinant HIN-1 as described (24). Membranes were washed and then exposed to film to detect bound HIN-1. Competition with 1,000x molar excess of cold HIN-1 eliminated binding and iodinated dermcidin (25), an unrelated secreted protein of similar size, did not bind these compounds (data not shown).

Cell proliferation, apoptosis, migration, and invasion assays. To determine the effect of HIN-1 expression on cell growth, we plated 4,000 cells per well in a 24-well plate and grew them in their standard growth media. In the case of the TET inducible cells, proliferation of the cells was compared in the presence and absence of doxycycline. Cells were counted (three wells per time point) on days 1, 3, 4, 7, 9, 11, 14, and 17 after plating. To determine the effect of HIN-1 on anchorage-independent growth, 40,000 cells per 25-cm2 flask were cultured in Noble agar at a final concentration of 0.3%. Colonies were counted after 21 days. For three-dimensional culture in Matrigel, we plated 40,000 cells suspended in 250 µl/well of Matrigel (BD Biosciences, San Jose, CA) in 24-well plates. Cells were counted after digestion of the Matrigel matrix with 2 units/mL dispase (Invitrogen, Carlsbad, CA) for 60 minutes at 37°C. For collagen growth, 7,500 cells were suspended in 1.5 mL/well of 1.2 mg/mL collagen I (Vitrogen 100, Cohesion Technologies, Palo Alto, CA) in 12-well plates. Cells were counted after digestion of the collagen matrix with 4 mg/mL collagenase (Sigma, St. Louis, MO) for 60 minutes at 37°C. Apoptosis (activated caspase 3) assays were carried out using the ApoTag kit (Promega, Madison, WI) following the manufacturer's instructions. In brief, MDA-MB-435S pBI-EGFP-HIN-1 cells were grown in the presence or absence of doxycycline at an initial density of 2 x 106 per 150-mm plate. As a positive control for apoptosis, an additional sample was grown in 0.2 µg/mL doxorubicin (Adriamycin). At the indicated time points, the cells were lysed in lysis buffer and the protein concentration determined using a Bio-Rad DC assay (Bio-Rad, Hercules, CA). Caspase 3 activity was then determined with the ApoTag kit reagents using 50 µg of protein/sample. To determine if HIN-1 expression influences cell migration and invasion, we tested the indicated cell types using BIOCOAT Matrigel invasion chambers (BD Biosciences, San Jose, CA), essentially as described (26). For invasion assays, we plated 2.5 x 104 cells per well and assayed 24 hours later; whereas for migration assays, we used 1.25 x 104 cells per well and determined cell numbers 12 hours later. The assays were done using serum-free medium supplemented with 1 mg/mL bovine serum albumin in the top chamber. In the bottom chamber, the same medium with 10% fetal bovine serum was used as the chemoattractant. All of the above-described experiments were repeated at least thrice.

[3H]bromodeoxyuridine incorporation and cell cycle. To determine DNA synthesis, we analyzed the incorporation of [3H]bromodeoxyuridine ([3H]BrdUrd) into the cells at various time points following release into the cell cycle in the presence or absence of HIN-1, essentially as described (27). Briefly, synchronized cells were replated into fresh medium, pulse labeled with 3 µCi [3H]BrdUrd for 1 hour followed by lysis in 0.1 mol/L NaOH and quantitation of [3H]BrdUrd incorporation in a scintillation counter. For cell cycle analysis, aliquots of cells were collected by trypsinization at the indicated times, washed with PBS, then fixed with an equal volume of ice-cold 80% ethanol added dropwise with vortexing, and stored at 4°C. Just before analysis, the samples were washed with PBS and then incubated with 2.5 µg/mL propidium iodide and 50 µg/mL RNase at 37°C for 30 minutes. Samples were then analyzed by flow cytometry.

Western blot analysis and AKT kinase assay. Cells were serum starved for 12 hours and then stimulated with epidermal growth factor (EGF, 1 µg/mL) or left untreated. Cells were washed with ice cold PBS and then incubated for 5 minutes in lysis buffer containing 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 1% NP40, 1 mmol/L NaF, 1 mmol/L Na4O7P2, protease inhibitor cocktail, and 1 mmol/L sodium orthovanadate. Cell lysates were collected by scraping off the plates, sonicated for 10 seconds, and centrifuged for 10 minutes at 14,000 rpm at 4°C. Protein concentration was determined using Bio-Rad detergent-compatible protein assay. Samples containing equivalent amounts of protein were resolved by NuPage 4 to 12% gel electrophoresis (Invitrogen) and transferred to polyvinylidene difluoride membrane. Immunoblotting was done using the following antibodies according to the manufacturer's instructions: anti-PDK1, anti-AKT, anti-phospho-AKT Ser473, anti-phospho-AKT Thr308, and anti-phospho-glycogen synthase kinase-3 (GSK-3) Ser21/9 (all from Cell Signaling, Beverly, MA). Signals were visualized using the Supersignal detection system (Pierce, Rockford, IL). AKT kinase assays were carried out using an AKT kinase kit and following the manufacturer's instruction (Cell Signaling).

p27 intracellular localization assays. MDA-MB-435S pBI-EGFP-HIN-1 cells (clone 26, 1.5 x 105) were seeded in six-well plates and incubated in the presence or the absence of doxycycline for 24 hours. Cells were then transfected using Fugene 6 (Roche, Indianapolis, IN) with phospho-EYFP expressing wild-type or T157A mutant p27 protein, both fused to yellow-green fluorescence protein (YFP). Cells were labeled with 2.5 µg/mL Hoechst 33342 to visualize nuclei. Localization of the fusion proteins and nuclei were detected by fluorescence microscopy.

HIN-1 methylation and epidermal growth factor receptor and PIK3CA mutational analysis. HIN-1 methylation-specific PCR analysis on lung and breast cancer DNA was done as described (2). Mutational analysis of EGF receptor (EGFR) in lung cancer specimens and PIK3CA in breast cancer specimens was done as described (28, 29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of HIN-1 on cell growth, migration, and invasion. To evaluate the effect of HIN-1 on breast cancer cell lines, we first used a tetracycline-regulated expression system (30, 31). Because our previous data suggested that HIN-1 expression suppresses cell growth (2), we employed the TET-OFF (activator represses expression in the presence of tetracycline) rather than the TET-ON (activator induces expression in the presence of tetracycline induction) inducible system. The TET-OFF approach minimizes negative selection against the expression plasmid and has been successfully used for the expression of known tumor suppressor genes (21). Stable clones of the human breast cancer cell line MDA-MB-435S were established with inducible expression of HIN-1 both with (clone 7) and without (clone 26) coexpression of EGFP. In clone 7, expression of EGFP is induced 6 hours following removal of doxycycline, whereas HIN-1 protein is detected in the medium ~18 hours later (Fig. 1A).



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  		Figure 1. Inhibition of cell growth, migration, and invasion by HIN-1 in MDA-MB-435S pBI-EGFP-HIN-1 cells. A, characterization of the time course of EGFP (top) and HIN-1 (bottom) expression following doxycycline removal in MDA-MB-435S pBI-EGFP-HIN-1 cells (clone 7). GFP and HIN-1 expression becomes detectable at 6 and 12 hours after removal of doxycycline, respectively, and further increases with time. B, growth curves of MDA-MB-435S pBI-EGFP-HIN-1 cells (two independent clones, clone 7 and clone 26) in the presence and absence of doxycycline. HIN-1 expression (–DOX) decreased cell growth in both clones. Representative experiment. Bars, SD. C, representative image of a soft agar assay done with MDA-MB-435S pBI-EGFP-HIN-1 cells (clones 26 and 7) cultured in the presence or absence of doxycycline (left). Expression of HIN-1 dramatically suppressed anchorage-independent growth and the invasive morphology. Quantitative summary of the result of a representative soft agar assay with colony counts plotted (right). Numbers reflect one representative experiment done in triplicate. The experiment was repeated thrice with the same result. D, three-dimensional growth of MDA-MB-435S pBI-EGFP-HIN-1 cells (clones 26 and 7) in Matrigel in the presence and absence of doxycycline (left). Similar to the soft agar assays, the expression of HIN-1 dramatically inhibited the growth of the cells and altered their morphology and growth pattern. HIN-1–expressing cells remained as small, tight clusters of cells, whereas the controls demonstrated extensive branching pattern indicative of invasive phenotype. Quantitative summary of three-dimensional growth assays done by plating the cells in Matrigel or collagen I (collagen growth), or on top of solidified collagen (collagen invasion; right). Numbers reflect one representative experiment done in triplicate. Bars, SD. The experiment was repeated thrice with identical results. E, HIN-1 expression inhibits cell migration and invasion MDA-MB-435S pBI-EGFP-HIN-1 cells. Cell migration and invasion was determined in the presence and absence of doxycycline (DOX) in two independent clones (clones 7 and 26). Y-axis, number of cells migrating or invading through the membrane per well. Representative experiment done in triplicate using clone 26. Repeated experiments and using both clones gave the same results.

 
The HIN-1 inducible MDA-MB-435S clones were used to characterize the effect of HIN-1 expression on cell growth, survival, migration, and invasion. Consistent with the growth inhibition initially observed in colony growth assays (2), HIN-1 expression resulted in marked suppression of cell growth under both exponential (Fig. 1B) and synchronized growth conditions (data not shown) As a control, treatment of the parental MDA-MB-435S cells with doxycycline alone had no effect on growth (data not shown). As anchorage-independent growth is a hallmark of the neoplastic phenotype, we also assessed the effect of HIN-1 expression on growth in soft agar. Again, expression of HIN-1 was associated with a dramatic inhibition of colony growth (Fig. 1C). Similar inhibition was observed when the MDA-MB-435S inducible cells were cultured in Matrigel or collagen I (Fig. 1D). Interestingly, in addition to decreasing colony number and size in these culture systems, induction of HIN-1 expression lead to striking morphologic changes (Fig. 1D). Although HIN-1–expressing cells remained as tight, round clusters of cells, the non-HIN-1 controls exhibited a branching, spiculated growth pattern characteristic of an invasive phenotype. This finding suggested that HIN-1 might influence invasive behavior. Thus, we next sought to determine whether the effects of HIN-1 on the invasive morphology of the MDA-MB-435S cells were due to direct effects of HIN-1 on the ability of the MDA-MB-435S to migrate or invade through extracellular matrix. Indeed, induction of HIN-1 expression led to a marked diminution of both migration and invasion in a Boyden chamber–type assay (Fig. 1E). Thus, consistent with a putative tumor suppressor role, induction of HIN-1 expression in a breast cancer cell line results in inhibition of anchorage-dependent and anchorage-independent growth, cell migration, invasion, and reversion of invasive morphology.

It is important to determine if the effects of HIN-1 expression observed using the inducible MDA-MB-435S cells are reflective of the physiologic function of HIN-1 and therefore generalizable, or are they specific to the particular expression system or cell line used in the assay. We were also interested in whether the effect of HIN-1 differed between cancer and nontransformed cell lines. To address these issues, two additional HIN-1 expression systems were developed: a recombinant adenovirus encoding the full-length human HIN-1 cDNA and an alkaline phosphatase-HIN-1 fusion protein (AP-HIN-1) that allowed us to test a larger set of cell lines. Expression of HIN-1 using the recombinant adenovirus inhibited growth of MDA-MB-435S and MCF-7 breast cancer cells and MCF-10A-immortalized mammary epithelial cells compared with cells infected with a LacZ-expressing control adenovirus (Fig. 2A). Similar results were seen in MDA-MB-468, MDA-MB-231, HCC1937 human breast cancer lines, HME50 human immortalized mammary epithelial cells, and H1355 and H157 human lung cancer cells (data not shown). In contrast, the HIN-1 adenovirus had no effect on the growth of T47-D human breast cancer cells and immortalized VA-13 fibroblasts despite evidence of substantial HIN-1 expression in those cells (data not shown).



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  		Figure 2. Effect of adenoviral-expressed HIN-1 and AP-HIN-1 on cell proliferation, migration, and invasion. A, expression of HIN-1 using a recombinant adenovirus (dashed line) inhibits cell growth in MDA-MB-435S, MCF-7, and MCF-10A cells. Adenovirus expressing LacZ (solid line) was used as control. Representative of multiple independent experiments showing the same result. B, exogenous addition of HIN-1 as a conditioned medium containing AP-HIN-1 (dashed line) leads to growth arrest in MCF-10A cells, whereas it has no effect on VA-13-immortalized fibroblasts and T47-D breast cancer cells. Conditioned medium containing AP alone (solid line) was used as a control. Based on our AP-HIN-1 binding studies, VA-13 fibroblasts do not have a receptor for HIN-1, whereas the mechanism underlying the resistance of T47-D cells to HIN-1–mediated growth arrest is currently unknown. C, expression of HIN-1 using a recombinant adenovirus in MDA-MB-435S cells inhibits cell migration and invasion. Adenovirus expressing LacZ was used as control. Y-axis, number of cells migrated or invaded through the membrane per well. Experiment was done thrice in triplicate. Bars, SD.

 
To determine if adenovirus-generated HIN-1 also affected the migratory and invasive capability of breast cancer cell lines, MDA-MB-435S and MDA-MB468 cells were infected with HIN-1 or control adenovirus and assessed for migration and invasion in Boyden chamber assays. Similar to results obtained in the inducible MDA-MB-435S system, HIN-1 adenovirus–infected cells showed markedly diminished migration and invasion in both breast cancer cell lines (Fig. 2C; data not shown). One hypothesis to explain the effect of HIN-1 on the migration, invasion, and morphology of breast cancer cell lines is that HIN-1 expression modulates the expression or function of extracellular matrix–degrading proteases. However, we observed no differences in protease expression by SAGE analysis or using a fluorochrome-based activity assay (data not shown).

To test the effects of soluble exogenous HIN-1 on cell growth, several cell lines were treated with conditioned medium containing the AP-HIN-1 fusion protein or control AP protein alone. Growth inhibition was observed in MCF-10A, HME50, H1355, H157, MDA-MB-435S, and MCF-7 cells exposed to the AP-HIN-1 fusion protein compared with control AP-treated cells (Fig. 2B; data not shown). Again, HIN-1 had no effect on the T47-D and VA-13 cell lines (Fig. 2B). In summary, in three distinct expression systems and across many different breast and lung cancer cell lines and also in immortalized mammary epithelial cells, HIN-1 expression resulted in pronounced suppression of cell growth, migration, and invasion.

Effect of HIN-1 on cell cycle progression and survival. To characterize the effects of HIN-1 on cell growth in greater detail, inducible HIN-1 MDA-MB-435S cells were synchronized in the G0 phase of the cell cycle, induced to express HIN-1, released into the cell cycle, and assessed for [3H]BrdUrd incorporation. Compared with controls not expressing HIN-1, the HIN-1-induced cells had greatly diminished incorporation of [3H]BrdUrd, suggesting that HIN-1 inhibits entry into the S phase (Fig. 3A). To further address HIN-1's effects on cell cycle progression, the inducible MDA-MB-435S were again synchronized in G0, released with or without HIN-1 induction, and analyzed for cell cycle progression by flow cytometry. Whereas control cells entered the S phase at 24 hours and subsequently continued through the cell cycle, cells expressing HIN-1 remained in G1-G0 and gradually entered a sub-G0 state, consistent with apoptosis (Fig. 3B). Interestingly, induction of HIN-1 expression in exponentially growing MDA-MB-435S cells resulted in cells accumulating in a sub-G0 state, but in contrast to that observed in the synchronized cells, no appreciable cell cycle arrest was detected (Fig. 3C). As phosphorylation of the retinoblastoma (Rb) protein is a hallmark of progression from G1 to S phase, we analyzed the effect of HIN-1 expression on Rb phosphorylation in the inducible MDA-MB-435S cells under synchronized and exponential growth conditions. Consistent with the flow cytometry results, HIN-1 expression inhibited Rb phosphorylation in synchronized but generally not in exponentially growing cells (Fig. 3D). This data thus offers further support for the hypothesis that HIN-1 expression can prevent cells in G0 from entering the cell cycle, whereas in exponentially growing cells, it has no apparent effect on cell cycle progression. Under both conditions, however, cells rapidly acquire sub-G0 DNA levels, suggesting that HIN-1 induces apoptosis. To explore this possibility, we assessed activated caspase 3 activity in MDA-MB-435S cells induced to express HIN-1. At both the 48-and 120-hour time points, the HIN-1–expressing cells showed substantially more caspase 3 activity than samples without HIN-1 expression (Fig. 3E). As a control for apoptosis and the assay, an additional sample without HIN-1 expression was treated with the chemotherapeutic agent doxorubicin (Adriamycin), a known inducer of apoptosis. This sample showed comparable caspase activity with the HIN-1–expressing cells (Fig. 3E). Together these data support the hypothesis that HIN-1 expression induces apoptosis in breast cancer cells, in addition to its inhibitory effect on the cell cycle progression of these cells.



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  		Figure 3. Effect of HIN-1 on cell cycle progression and survival in MDA-MB-435S pBI-EGFP-HIN-1 cells. A, HIN-1 expression in synchronized cells inhibits [3H]BrdUrd incorporation (H3 BrDU). Cells were synchronized by serum deprivation and confluence and pulse labeled with [3H]BrdUrd at the indicated times following release into serum containing medium. The majority of HIN-1–expressing cells (dashed line) did not take up ([3H]BrdUrd) and thus did not enter the S phase compared with controls (solid line). Representative experiment repeated thrice with the same results. B, flow cytometric analysis of cell cycle progression in control and HIN-1–expressing cells. MDA-MB-435S pBI-EGFP-HIN-1 cells were synchronized by confluence and serum deprivation. Cell cycle reentry was induced by plating the cells at low cell density into fresh medium containing 10% serum. HIN-1 expression was induced 24 hours before release and was maintained throughout the experiment. Cells were collected at the indicated time points after release and labeled with propidium iodide. Control cells entered the S phase at ~24 hours after replating, whereas HIN-1–expressing cells remained in G0-G1 phase and eventually underwent apoptosis. C, induction of HIN-1 expression in exponentially growing in MDA-MB-435S pBI-EGFP-HIN-1 cells leads to the accumulation of sub-G1 cells suggesting the induction of apoptosis, whereas no apparent cell cycle arrest is detected. D, Western blot analysis of Rb protein phosphorylation (Ser807/Ser811) in control and HIN-1–expressing cells. The status of Rb phosphorylation was determined by immunoblot analysis using cell extracts prepared at the indicated time points following cell cycle reentry in synchronized and exponentially growing cells, respectively. Correlating with the results of flow cytometric analysis, HIN-1 expression inhibits Rb phosphorylation in synchronized but generally not in exponentially growing cells. E, measurement of activated caspase 3 activity in control (DOX+, black columns) and HIN-1–expressing (DOX–, white columns) cells at 48 and 120 hours after HIN-1 induction. MDA-MB-435 cells treated with 0.2 µg/mL doxorubicin (Adriamycin) were used as control (ADRIA, gray column). Bars, SD.

 
Mechanism of HIN-1–mediated growth inhibition. To begin to dissect the molecular mechanism of HIN-1–mediated growth arrest and apoptosis, we used SAGE to analyze changes in the global gene expression profile of exponentially growing MDA-MB-435S cells following the induction of HIN-1. Samples of mRNA were obtained from these cells before and 24 and 48 hours after induction of HIN-1 expression. Surprisingly, besides HIN-1 itself, we did not identify any transcripts that were statistically significantly differentially expressed between control and HIN-1 expressing cells (data not shown; SAGE libraries deposited into SAGE Genie database),7 suggesting that transcriptional changes may not play a role in HIN-1–mediated apoptosis in exponentially growing cells, at least not at the early time points examined. We therefore analyzed the involvement of known signaling pathways in HIN-1 action using activated state (phosphorylation) specific antibodies for various kinases and other signaling proteins. Our data showing that HIN-1 expression inhibits cell cycle reentry suggested that HIN-1 may antagonize mitogenic signaling pathways necessary for cell cycle reentry. Thus, first, we screened for changes in response to EGF treatment in the presence or absence of HIN-1 expression. We found that the phosphorylation of AKT at Ser473 was modestly inhibited and decreased more rapidly in MDA-MB-435S cells preinduced to express HIN-1 relative to control MDA-MB-435S cells not expressing HIN-1, whereas the level of total AKT was not affected by HIN-1 expression (Fig. 4A). Similar results were noted in parental MDA-MB-435S and MCF-7 cells treated with EGF and medium containing AP or AP-HIN-1. Phosphorylation of AKT was induced later and at lower levels in both cell lines treated with AP-HIN-1 (Fig. 4B) indicating that the observed effects of HIN-1 on AKT phosphorylation are not restricted to the TET inducible system. Importantly, although AP-HIN-1 inhibited EGF-mediated AKT phosphorylation in MDA-MB-435S and MCF-7 cells, both of which showed growth inhibition in response to AP-HIN-1, no effect of AP-HIN-1 on AKT phosphorylation was noted in T47-D, HCC1937, and MDA-MB468 cells, lines whose growth was not affected by AP-HIN-1 (Fig. 4B). Interestingly, adenoviral overexpression of HIN-1 inhibited the growth of MDA-MB-468 and HCC1937 but not T47-D cells. The differing effects of AP-HIN-1 and adenoviral HIN-1 on the growth of MDA-MB-468 and HCC1937 is likely to be due to the different concentration of HIN-1 protein in the medium in the two conditions. Thus, MDA-MB-468 and HCC1937 may be less sensitive but not completely refractory to HIN-1 and could be inhibited when it is present at high local concentrations such as achieved by adenoviral overexpression. MDA-MB-468 and HCC1937 cells have homozygous deletion of PTEN resulting in constitutively active AKT that may lead to their partial HIN-1 resistance, whereas the molecular basis of the resistance of T47-D cells to HIN-1 could potentially be due to the recently reported amplification of PI3KCA in these cells (32). These data support the hypothesis that down-regulation of the AKT pathway is involved in HIN-1–mediated growth inhibition. Because HIN-1 is a secreted protein, we postulated that one mechanism by which HIN-1 could inhibit EGF-induced activation of AKT was by sequestering extracellular EGF and preventing its binding to EGFR. However, attempts to coimmunoprecipitate HIN-1 and EGF failed to detect any interaction between the two proteins in culture media (data not shown).



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  		Figure 4. Effect of HIN-1 on AKT phosphorylation and kinase activity and p27 localization. A, serum-deprived MDA-MB-435S pBI-EGFP-HIN-1 cells were left untreated (–) or were induced to express HIN-1 (+) for 24 hours followed by stimulation of the cells with EGF. Cells were collected at the indicated time points following EGF treatment and AKT phosphorylation was analyzed by Western blot. The phosphorylation of AKT by EGF was slightly inhibited and decreased faster in HIN-1–expressing cells compared with controls, whereas total AKT protein levels are the same. B, breast cancer cell lines that are growth inhibited by HIN-1 (yes) or not growth inhibited by HIN-1 (no) were serum deprived and treated with EGF and medium containing AP (–) or AP-HIN-1 (+). In cells that are growth inhibited by HIN-1 (MDA-MB-435S and MCF-7), a decrease in AKT phosphorylation was detected following AP-HIN-1 treatment, whereas in cells that did not respond to AP-HIN-1 (T47-D and MDA-MB-468), there is no effect on AKT phosphorylation in the presence of HIN-1. C, AKT kinase activity in MCF-7 cells in the absence of serum and following serum stimulation in the presence of AP-HIN-1 or control AP conditioned medium. AKT kinase activity is dramatically decreased in AP-HIN-1-treated cells compared to controls. D, MDA-MB-435S pBI-EGFP-HIN-1 cells (clone 26) were incubated in the presence (+) or absence (–) of doxycycline for 24 hours before transfection with wild-type p27 or its T157A mutant fused to YFP. Intracellular localization of the fusion proteins was quantitated and expressed as % transfected (green) cells. Open columns, cytoplasmic localization; closed columns, nuclear p27-YFP fusion protein. Columns, average of triplicate wells from a representative experiment repeated twice; bars, SD. Right, representative images of the cells treated and transfected as described above; nuclei were visualized with Hoechst 33342 counterstaining (blue signal).

 
To determine if inhibition of AKT phosphorylation by HIN-1 affects AKT kinase activity, we did an in vitro AKT kinase assay using cell extracts prepared from MCF-7 cells stimulated with serum in the presence of AP-HIN-1 or control AP conditioned medium. A dramatic decrease in AKT kinase activity was observed in the AP-HIN-1-treated MCF-7 cells compared with controls (Fig. 4C). One mechanism by which activated AKT contributes to resistance to antiproliferative signals and promotes cell cycle progression is by phosphorylating p27 at Thr157 within its nuclear localization signal, resulting in impaired nuclear import and cytoplasmic accumulation of p27 (25). To determine if HIN-1 expression affects this important function of AKT in vitro, we transfected the TET inducible MDA-MB-435S cells with constructs expressing wild-type or T157A mutant p27 fused to YFP (33). Although the wild-type p27 fusion protein was predominately cytoplasmic in the absence of HIN-1, induction of HIN-1 expression resulted in the majority of p27 localizing to the nucleus, consistent with inhibition of AKT activity (Fig. 4D). As expected, the mutant p27 fusion protein, which cannot be phosphorylated, localized to the nucleus independent of HIN-1 expression (Fig. 4D).

HIN-1 and the phosphoinositide 3-kinase/AKT pathway. To further test the hypothesis that HIN-1–mediated growth inhibition involves the down-regulation of the AKT pathway, we generated pools of MCF-7 and MDA-MB-435S cells that overexpress myrAKT, a constitutively active form of the kinase. The overexpression and activity of myrAKT was confirmed by immunoblot analysis of phospho-AKT and one of its substrates, phospho-GSK3ß (Fig. 5A). To determine the effect of the constitutively active AKT on HIN-1–mediated growth arrest, the proliferation of myrAKT-expressing and control cells was compared after infection with HIN-1 or control ß-galactosidase (LacZ) expressing adenovirus. Although the control cells, as expected, showed decreased growth in response to HIN-1 expression, this growth inhibition was completely abrogated in the myrAKT-expressing MCF-7 and MDA-MB-435S cells (Fig. 5B). MyrAKT overexpression did not influence the presence of the putative HIN-1 receptor on the cell surface nor its binding of HIN-1 (data not shown). This data provides further evidence that the AKT signaling pathway is involved in HIN-1–mediated growth inhibition.



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  		Figure 5. Overexpression of constitutively activate AKT abrogates HIN-1–mediated growth arrest. A, characterization of stable pools of control, neomycin-resistant (N) and myrAKT (A) expressing MCF-7 and MDA-MB-435S cells. The expression and constitutive activity of myrAKT was confirmed by immunoblot analysis of phospho-AKT (pAKT) and phospho-GSK3ß (pGSK3ß) in serum-deprived and growth factor–deprived cells. No or very low phospho-AKT and phospho-GSK3ß is detected in control neomycin cells, whereas AKT and GSK3ß were constitutively phosphorylated in myrAKT-expressing cells. B, growth curves of neomycin resistant (Neo) and myrAKT expressing MCF-7 and MDA-MB-435S cells following infection with a recombinant adenovirus expressing ß-galactosidase (Ad-LacZ, solid line) or HIN-1 (Ad-HIN-1, dashed line). Cells were infected with Ad-LacZ or Ad-HIN-1 adenoviruses, and cell numbers were determined at the indicated times after infection. Expression of HIN-1 inhibited the growth of control, neomycin-resistant MCF-7, and MDA-MB-435S cells, whereas it had no effect on cells expressing constitutively active AKT (myrAKT). Representative experiment done in triplicate and repeated at least thrice with the same results. Bars, SD.

 
To begin to determine at what point in the AKT signaling pathway HIN-1 acts, we attempted to generate clones of the MDA-MB-435 and MCF-7 cell lines that overexpress a constitutively active form of the catalytic domain of phosphoinositide-3-kinase (PI3KCA), using a recombinant retroviral system. Unfortunately, despite multiple attempts using several different constructs expressing wild-type or mutant PI3KCA, we could not derive overexpressing clones. Other groups have had similar experience working with PI3K in breast cancer cell lines,8 suggesting that increased PI3K activity may be toxic to some breast cancer cells. We were able to generate clones of MCF-7 and MCF-10A cells that overexpress 3-phosphoinositide-dependent kinase 1 (PDK1), a kinase directly involved in AKT activation via phosphorylation of Thr308 of AKT1. However, in contrast to the results observed with overexpression of activated AKT (Fig. 5B), overexpression of PDK1 had no effect on HIN-1–mediated growth inhibition in either cell line (data not shown). Further analysis of these PDK1-overexpressing clones showed that despite marked overexpression of PDK1 protein in these cell lines, there was no evidence of constitutive AKT activity in the absence of serum or enhanced AKT phosphorylation in response to EGF (data not shown). This result is consistent with the hypothesis that prevention of HIN-1–mediated growth inhibition requires constitutive AKT activation.

Several recent studies have identified activating mutations in the catalytic subunit of PI3K (PI3KCA) in a substantial percentage of human breast cancers (28, 34–36). Based on the data presented above, strongly suggesting that HIN-1–mediated growth arrest is mediated through the AKT signaling pathway, we postulated that there may be an interaction between a tumor's HIN-1 expression status and the presence or absence of PI3KCA mutations. Because hypermethylation of the HIN-1 promoter is the primary mechanism by which HIN-1 expression is silenced in breast cancers, we used HIN-1 methylation as a surrogate for HIN-1 expression. In a set of 114 primary breast cancers, HIN-1 methylation occurred in a greater percentage of tumors with PI3KCA mutations (19 of 30 or 63%) than in tumors without such mutations (40 of 93 or 43%). Although this association was of borderline statistical significance (P = 0.06, Fisher exact test) and needs to be repeated in a larger set of tumors, it suggests that the selective pressure to down-regulate HIN-1 expression may be stronger in cancers that have an activated PI3K/AKT pathway. In light of our finding that HIN-1 inhibits EGF-mediated AKT activation (Fig. 4A), we also examined the HIN-1 methylation and EGFR mutation status of a series of 74 adenocarcinomas of the lung. In this series, there was no association between EGFR mutations and HIN-1 methylation. Seven of 31 samples (23%) and 10 of 43 samples (23%) with mutant and wild-type EGFR, respectively, had methylated HIN-1 (P = 1.00, Fisher exact test).

Identification of a putative HIN-1 receptor in vitro and in vivo. Conclusive evidence indicates that HIN-1 is a secreted protein and acts extracellularly: (a) the HIN-1 cDNA encodes a protein with a NH2-terminal secretory signal peptide; (b) HIN-1 protein can be isolated from the medium of HIN-1–expressing cells and from human saliva, bronchial fluid, and blood (7); (c) A soluble AP-HIN-1 fusion protein is capable of recapitulating the inhibitory effects of endogenous HIN-1 on cell growth and AKT phosphorylation; and (d) overexpression of HIN-1 without its signal peptide has no effect on colony cell growth (data not shown). We therefore sought to identify a putative cell surface receptor through which HIN-1 mediates its effects. Using a recombinant AP-HIN-1 fusion protein, we did ligand-binding assays in vitro using various cell lines and in vivo using frozen sections of normal and cancerous breast and lung tissue. As depicted in Fig. 6A, MCF-10A human mammary epithelial cells but not VA-13 fibroblasts show specific cell surface binding of HIN-1. Several other breast cancer cell lines including MD-MB-435, MCF-7, and T47-D cells also seem to specifically bind HIN-1 on their cell surface (Fig. 6A). Similarly, staining of human normal and cancerous breast and lung tissue with AP-HIN-1 indicates specific binding to normal luminal mammary and bronchial epithelial cells as well as to breast cancer cells (Fig. 6B). In general, the presence of cell surface HIN-1 binding correlates with the cellular response to HIN-1 further strengthening that its action is receptor mediated. For example, VA13 fibroblasts do not seem to have specific cell surface HIN-1 binding and are not growth inhibited by HIN-1. Because HIN-1 is highly expressed in normal luminal mammary and bronchial epithelial cells, the finding that the same cell type expresses the candidate HIN-1 receptor suggests that HIN-1 acts as an autocrine factor. To further analyze HIN-1 binding to its putative receptor, we did Scatchard plot analysis in MCF-10A human mammary epithelial and VA13 human fibroblast cells (Fig. 6C). Based on this assay, MCF-10A cells seem to express a saturable HIN-1 binding protein that binds HIN-1 with an estimated KD = 2.8 x 10–9 mol/L, which is in the range of typical high-affinity receptor-ligand interactions. In addition, AP-HIN-1 binding was decreased following preincubation of the cells with conditioned medium from HIN-1–expressing cells or with purified recombinant HIN-1 protein (data not shown), indicating the specificity of the binding. Based on these binding studies, we estimate the presence of ~40,000 HIN-1 receptors on the cell surface.



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  		Figure 6. Identification of a putative HIN-1 receptor. A, binding assays done with AP-HIN-1 in the indicated breast cell lines, organoids (primary cultured breast epithelial cells), COS7 cells, and VA-13 fibroblasts. Y-axis, specific alkaline phosphatase activity (calculated as AP-HIN-1 activity – AP control). B, in situ staining for the putative HIN-1 receptor in breast and normal lung tissue. Sections of breast tissue containing both normal (N) and tumor (T) epithelium and lung tissue with bronchi were incubated with AP control (middle) or AP-HIN-1 fusion protein (bottom). Bound AP forms an insoluble blue precipitate upon addition of substrate. AP-HIN-1 specifically bound to normal and cancerous mammary epithelial cells in the breast and bronchial epithelial cells in the lung indicating the presence of HIN-1 binding activity. Arrows point to dark blue cells with strong AP-HIN-1 binding. C, Scatchard analysis of AP-HIN-1 binding to MCF-10A-immortalized mammary epithelial cells and VA-13 fibroblasts. MCF-10A cells or VA-13-transformed human fibroblasts were treated with varying amounts of AP-HIN-1 and then tested for bound AP activity (top). The same data for MCF-10A cells presented as a Scatchard plot (bottom) that suggests a presence of one high-affinity (KD = 2.8 x 10–9 mol/L) binding slope for HIN-1 on MCF-10A epithelial cells. The specificity of the interaction was further confirmed by using excess untagged HIN-1 or an irrelevant secreted protein as competitor. No specific HIN-1 binding was detected on the surface of VA-13 fibroblasts.

 
Uteroglobin was first identified as a lipid-binding protein potentially playing a role in uterine implantation by sequestering hormones such as progesterone (37). Subsequently, uteroglobin was shown to bind several other compounds including polychlorinated biphenyl metabolites (38). Because of the structural homology between HIN-1 and uteroglobin, we explored whether HIN-1 had similar lipid binding properties. Based on ligand blot assays, we detected strong binding of HIN-1 to progesterone, tri-iodo-thyronine, as well as a 30-amino-acid synthetic peptide derived from HIN-1 (data not shown). The physiologic relevance of HIN-1 binding to these compounds is unclear, whereas HIN-1 association with itself (dimerization and formation of higher molecular weight oligomers) has been observed using other assays and sources of HIN-1 protein. Recent studies suggest that uteroglobin may also bind to cubilin and the cubilin/megalin receptor complex may play a role in the internalization of uteroglobin (39). To test if similar to uteroglobin HIN-1 may also bind cubilin, we tested the binding of recombinant HIN-1 to cubilin using a surface plasmon resonance (Biacore) assay but found no evidence of specific high-affinity binding (data not shown). The molecular identification and characterization of the HIN-1 signaling receptor is currently in progress.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we report the functional characterization of the HIN-1 putative tumor suppressor gene. Despite extensive screening, we were unable to identify a cell line with endogenous expression of HIN-1 that could be used for loss-of-function (e.g., RNA interference) studies. We therefore used an inducible expression system as well as adenoviral and fusion protein systems to determine the effects of HIN-1 on breast cell lines. Using all three approaches, we found that HIN-1 dramatically suppresses both anchorage-dependent and anchorage-independent growth of these cells. This inhibition is a combined result of induction of apoptosis and prevention of cell cycle reentry of growth-arrested cells. HIN-1 also suppresses cell migration and invasion, leading to a reversion of the invasive morphology of cancer cell lines in three-dimensional culture. Importantly, these effects were observed in multiple cell lines, both transformed and nontransformed, and were similar regardless of whether endogenous HIN-1 (TET inducible system and HIN-1–expressing adenovirus) or exogenous HIN-1 (HIN-1 fusion protein) was used. This uniformity of response across several model systems strongly suggests that the observed effects reflect actual physiologic functions of HIN-1.

HIN-1 has distant homology to uteroglobin, the prototypical member of the secretoglobin superfamily of proteins (9, 10). Despite the rather low level of homology between uteroglobin and HIN-1, our data indicate that the two proteins seem to share quite similar functions. Both inhibit many features of the invasive phenotype, including anchorage-dependent and a7nchorage-independent growth, and invasion of extracellular matrix (12–15). HIN-1 and uteroglobin ligand binding activity, suggesting putative receptor expression, exists on the same cell types that express these proteins, suggesting that they act in an autocrine manner. Uteroglobin knockout mice are prone to both spontaneous and chemically induced cancers (16, 17). Preliminary data suggests that mice with homozygous deletion of HIN-1 also are predisposed to develop spontaneous malignancies.9 In addition, expression of both proteins is frequently lost in several types of epithelial cancers and in the case of HIN-1, loss of expression is due to promoter hypermethylation and is associated with poor clinical outcome (5). Taken together, these data strongly suggest that HIN-1 and uteroglobin define a new class of secreted proteins with tumor suppressor function.

Despite the extensive data describing the tumor suppressor–like functions of uteroglobin, little is known about its mechanism of action. There is evidence that uteroglobin has a role in binding progesterone and other lipophilic ligands, and this function may be mediated via cubilin and its endocytic partner, megalin (39). However, it is unclear if this lipophylic compound binding activity and internalization by cubilin/megalin accounts for the tumor suppressor effects of uteroglobin. Additional high-affinity binding sites for uteroglobin have been described, but the identity of these putative receptors is unknown (15). Similarly, HIN-1 seems to bind progesterone and it has been reported to specifically interact with MARCO, a class A scavenger receptor (40). MARCO binds bacteria and has an important role in the clearance of pathogens. It has no known signal transduction activity and therefore, like uteroglobin, the pathway through which the tumor suppressor–like effects of HIN-1 are transduced is unknown. Our observation that HIN-1 expression prevented cell cycle reentry suggested that HIN-1 may act by blocking mitogen-induced signal transduction, and in fact, we observed that HIN-1 expression inhibited the AKT phosphorylation induced by EGF. Further studies showed that HIN-1–mediated growth inhibition did not occur in all cell types. Specifically, responsiveness to HIN-1 correlated with the presence of a HIN-1 cell surface receptor and the ability of HIN-1 to suppress AKT phosphorylation. Moreover, HIN-1 inhibited AKT kinase activity and AKT's ability to suppress nuclear localization of p27 and forced expression of a constitutively active AKT abrogated HIN-1–mediated growth inhibition. These data provide strong support for the hypothesis that HIN-1–mediated growth inhibition occurs via modulation of AKT signaling. In addition, a recent report describing the induction of HIN-1 expression by EGF in mouse lung epithelial cells provides further evidence linking HIN-1 with mitogen-induced signaling pathways (41).

In summary, we show here that HIN-1 inhibits cell cycle reentry, suppresses migration and invasion, and induces apoptosis in breast cell lines. Substantial evidence indicates these effects are mediated by a high-affinity cell surface receptor and involve the modulation of the AKT signaling pathway. These data, together with prior results showing that HIN-1 expression is frequently lost in many common tumor types, and that this loss is associated with poor outcome in lung cancer, strongly suggest that elimination of HIN-1 function is an important event in tumorigenesis.


   Acknowledgments
 
Grant support: National Cancer Institute grant RO1 CA94074 (K. Polyak), K12 training grant 5K12CA87723-03 (I. Krop), and Department of Defense Postdoctoral fellowship grant DAMD17-02-1-0363 (D. Porter and M.T. Parker).

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 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Soren K. Moestrup (University of Aarhus, Aarhus, Denmark) for doing the Biacore binding assays; Drs. Joyce Slingerland, Jean Zhao, Tom Roberts, William Hahn, and William Sellers (DFCI) for providing reagents and experimental protocols; and Dr. Andrea Richardson, Gabriela Lodeiro, and Ruth Gomes (Brigham and Women's Hospital, Boston, MA) for help with the acquisition of human tissue samples.


   Footnotes
 
Note: I. Krop and M. Taylor Parker contributed equally to this article.

7 http://cgap.nci.nih.gov/SAGE. Back

8 J. Zhao, personal communication. Back

9 K. Polyak, unpublished data. Back

Received 5/16/05. Revised 8/ 8/05. Accepted 8/31/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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