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J Thorac Cardiovasc Surg 2007;133:733-737
© 2007 The American Association for Thoracic Surgery

General Thoracic Surgery

Wnt inhibitory factor inhibits lung cancer cell growth

Jae Kim, MD, Liang You, MD, PhD, Zhidong Xu, PhD, Kris Kuchenbecker, BS, Dan Raz, MD, Biao He, PhD, David Jablons, MD*

Thoracic Oncology Laboratory, UCSF Comprehensive Cancer Center, San Francisco, Calif.

Read at the Eighty-sixth Annual Meeting of The American Association for Thoracic Surgery, Philadelphia, Pa, April 29-May 3, 2006.

Received for publication May 22, 2006; revisions received September 15, 2006; accepted for publication September 29, 2006.

* Address for reprints: David Jablons, MD, Box 1724, 2200 Post St, Mount Zion Building C, University of California, San Francisco, San Francisco, CA 94143. (Email: jablonsd{at}surgery.ucsf.edu).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: Aberrant activation of the Wnt signaling pathway is associated with the pathogenesis of multiple cancers, including non–small cell lung cancer. Wnt inhibitory factor, a secreted Wnt antagonist, is downregulated in non–small cell lung cancer. We hypothesized that restoration of Wnt inhibitory factor function would inhibit lung cancer cell growth.

Methods: The lung cancer cell lines A549 and H460 were transfected with an expression vector containing the Wnt inhibitory factor gene. Apoptosis rates and colony formation were measured after transfection. Recombinant Wnt inhibitory factor protein was used to treat H460 cells, and proliferation rates were measured with an MTS assay. Finally, Wnt inhibitory factor plasmid was peritumorally injected near H460 tumor xenografts in nude mice.

Results: Wnt inhibitory factor–transfected cells had increased apoptosis and decreased colony formation than control cells. Recombinant human Wnt inhibitory factor protein was also able to inhibit H460 cell proliferation measured by using the MTS assay. Wnt inhibitory factor plasmid significantly inhibited the growth in vivo of H460 tumor xenografts in nude mice.

Conclusion: These data suggest that Wnt inhibitory factor is able to inhibit lung cancer cell growth both in vitro and in vivo and provides additional evidence that Wnt inhibitory factor plays an important role in Wnt pathway regulation in lung cancer.

Abbreviations and Acronyms RT-PCR = reverse transcription–polymerase chain reaction; WIF-1 = Wnt inhibitory factor


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
As the leading cause of cancer death worldwide, lung cancer poses a major public health problem. Non–small cell lung cancer (NSCLC) comprises 75% to 80% of lung cancer cases.1Go Despite recent advances in treatment, overall prognosis remains poor, and the overall 5-year survival rate remains at 15%.2Go A better understanding of the molecular pathogenesis of lung cancer might lead to more effective targeted therapies.

The Wnt signaling pathway is a key developmental pathway that has also been implicated in oncogenesis.3,4Go It plays an important role in normal embryonic and stem cell development but is dormant in most adult tissues. Aberrant reactivation of the Wnt pathway has been shown in multiple human cancers, including NSCLC.5-10Go

The Wnt pathway is regulated by the complex interplay of multiple agonists and antagonists. One such antagonist is Wnt inhibitory factor (WIF-1).

WIF-1 is a naturally occurring secreted protein that binds the Wnt ligand.11Go Downregulation of WIF-1 has been shown in multiple cancers.12-14Go We recently reported that WIF-1 was downregulated and silenced by promoter hypermethylation in NSCLC.15Go

We hypothesized that restoration of WIF-1 function would inhibit the Wnt pathway and thus inhibit lung cancer cell growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
NSCLC cell lines (NCI-H460 and A549) were obtained from American Type Culture Collections (Manassas, Va). NSCLC cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL).

Transfection
The human WIF-1 open reading frame was cloned into a mammalian expression vector, pcDNA3.1, obtained from Invitrogen (Carlsbad, Calif). Twenty-four hours before transfection, 2 x 105 cells (H460 or A549) were plated in 6-well plates. Eight micrograms of WIF-1 plasmid was transfected per well by using LipofectAMINE 2000 (Invitrogen), according to the manufacturer’s protocol. Eight micrograms of pcDNA3.1 vector containing the lac-Z gene (Invitrogen) was transfected into cells as a control.

Semiquantative Reverse Transcription–Polymerase Chain Reaction
Seventy-two hours after transfection, H460 and A549 cells were treated with trypsin and harvested. Total RNA from was isolated with an RNeasy Mini Kit (Qiagen, Valencia, Calif), according to the manufacturer’s protocol. Reverse transcription–polymerase chain reaction (RT-PCR) was performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, Calif) by using an RT-PCR kit (SuperScript One-step RT-PCR with Platinum Taq kit, Invitrogen) for 25 cycles, according to the manufacturer’s protocol. Primers for RT-PCR were obtained from Operon Technologies, Inc (Alameda, Calif). Primer sequences for the human WIF-1 cDNA were 5'-CCGAAATGGAGGCTTTTGTA-3' (forward) and 5'-TGGTTGAGCAGTTTGCTTTG-3' (reverse), primers for c-MYC were 5'-TTCGGGTAGTGGAAAACCAG-3' (forward) and 5'-CAGCAGCTCGAATTTCTTCC-3' (reverse), and primers for cyclin D1 were 5'-CTGTCGCTGGAGCCCGTGAA-3 27 (forward) and 5'-TGGCACAGACCCGAACGAAG-3' (reverse). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. RT-PCR products were then electrophoresed on an agarose gel and photographed under UV light.

Apoptosis Assay
Three days after transfection, cells were harvested after treatment with trypsin. They were then stained with an Annexin V FITC Apoptosis Detection Kit (Invitrogen Biosource, Carlsbad, Calif), according to the manufacturer’s protocol. Then stained cells were immediately analyzed by means of flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, NJ).

Colony Formation Assay
Transfection was performed as above. Twenty-four hours after transfection, cells were stripped and plated on 6-well cell-culture dishes. The cells were then selected by using G418 (400 µg/mL). Two weeks after selection, colonies were stained with 0.5% crystal violet solution and counted.

Recombinant WIF-1 and Cell Proliferation Assay
Purified recombinant human WIF-1 protein was obtained from R&D Systems (Minneapolis, Minn). The protein was resuspended in phosphate-buffered saline to obtain concentrations of 1 µg/µL.

H460 cells were plated at 5 x 103 cells per well in RPMI medium in a 96-well culture plate. Twenty-four hours after plating, half the cells were treated with recombinant WIF-1 protein, and half were treated with bovine serum albumin (BSA) (Sigma-Aldrich, St Louis, Mo) to achieve a concentration of 20 µg/mL. Medium was changed after 96 hours, at which point WIF-1 or BSA was again added to achieve concentrations of 20 µg/mL. The percentage of live cells was evaluated by incubating for 2 hours with the Celltitre 96 Aqueous Non-radioactive Cell Proliferation Assay (Promega, Madison, Wis). Plates were read with a spectrophotometer at a wavelength of 490 nm. Measurements were made at 3 time intervals: 24 hours after treatment, 96 hours after treatment, and 192 hours after treatment. Three wells from each treatment group were measured for each time point.

Tumor Xenografts
All in vivo experiments were performed in accordance with the University of California, San Francisco institutional guidelines (institutional review board A8714-25971-01). Groups of 8 female athymic nude mice (strain NCRNU-M, 5-6 weeks old) received subcutaneous injections with 1 x 107 H460 cells in the dorsal area at a volume of 100 µL. Three days later, the tumors were uniformly formed. We then subcutaneously injected the mice twice weekly in the peritumoral area with either 50 µg of WIF-1 vector or empty pcDNA3.1 vector in 100 µL of lipofectamine. Tumor size was determined at weekly intervals for 3 weeks, and tumor volume was calculated by using width (x) and length (y; [x2y/2, where x if less than y]). After the mice were killed, tumors were resected and weighed.

Statistical Analysis
The data shown represent mean values ± standard deviation. The unpaired t test was used to compare different treatments and determine the P value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previously, we found that WIF-1 was underexpressed in the NSCLC cell lines H460 and A549.15Go An expression vector containing the WIF-1 gene was transfected into H460 and A549 cells to test whether WIF-1 can inhibit growth and induce apoptosis in these cell lines. Vector containing lac-Z was used as a control. We confirmed increased WIF-1 expression in the WIF-1–treated cells by performing semiquantitative RT-PCR on RNA extracted from WIF-1– and lac-Z–transfected cells (Figure 1).


Figure 1
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  		Figure 1. Reverse transcription–polymerase chain reaction for Wnt inhibitory factor (WIF-1) in A549 and H460 cells transfected with either lac-Z (control) or WIF-1. The fragment of human WIF-1 cDNA amplified is 188 bp.

 
We confirmed downregulation of the Wnt pathway by measuring expression of the Wnt target genes c-MYC and cyclin D1 by using semiquantitative RT-PCR. We found that lung cancer cells transfected with WIF-1 had decreased expression of c-myc and cyclin D1 (Figure 2). Transcription of both signals has been shown to be mediated by ß-catenin through the TCF binding site.16-18Go


Figure 2
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  		Figure 2. Reverse transcription–polymerase chain reaction for cyclin D1 and c-myc in A549 and H460 cells transfected with either lac-Z (control) or Wnt inhibitory factor (WIF-1). Glyceraldehyde-3-phosphate dehydrogenase was used as a control transcript.

 
Three days after transfection, we found significantly increased apoptotic cell death in cells transfected with WIF-1 (P < .05, Figure 3). Having found increased apoptosis in WIF-1–transfected cells, we then assessed the effect of WIF-1 on growth inhibition. We selected for transfected cells and found that after selection, cells transfected with WIF-1 formed dramatically fewer colonies than did cells transfected with control vector (Figure 4).


Figure 3
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  		Figure 3. Annexin V analysis of apoptosis. Seventy-two hours after transfection with either lac-Z (control) or Wnt inhibitory factor (WIF-1), A549 and H460 cells were harvested by means of trypsinization and stained with an Annexin V-fluorescein isothiocyanate kit. The bar graphs show the percentage of apoptosis, which represents the number of Annexin V+ cells out of the total number of cells counted (mean ± standard deviation, P < .05).

 

Figure 4
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  		Figure 4. Colony formation assay with A549 and H460 cells. The cells were transfected with lac-Z or Wnt inhibitory factor (WIF-1) vector and selected with G418. Colonies were stained by using 0.5% methylene blue and counted 2 weeks after the transfection.

 
We then used recombinant human WIF-1 to validate the effect of WIF-1 in NSCLC cells. Purified recombinant human WIF-1 protein was added to the medium of H460 cells plated in 96-well plates. We used an MTS assay to assess cell proliferation at 24 hours, 96 hours, and 192 hours after treatment. We found that recombinant human WIF-1 significantly inhibited cell growth after 8 days of incubation (Figure 5).


Figure 5
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  		Figure 5. MTS assay of H460 cells treated with recombinant Wnt inhibitory factor (WIF-1) or BSA for 8 days. A, Proliferation was measured with the MTS assay at 3 time points: 1 day, 4 days, and 8 days after treatment. Absorbance was measured at 490 nm 2 hours after addition of MTS reagent. B, MTS assay after 8 days of treatment. Absorbance at 490 nm is expressed as percentage proliferation, where control treatment (BSA) is 100% (mean ± standard deviation, P < .05).

 
Finally, we assessed the effect of WIF-1 transfection in an in vivo tumor xenograft model. Nude mice were subcutaneously injected with H460 cells and then treated with peritumoral injections of WIF-1 plasmid or empty vector. We found the WIF-1 plasmid significantly inhibited tumor growth (Figure 6, A). After 4 weeks of treatment, the tumors treated with the control weighed more than 3 times as much as tumors treated with WIF-1 (P < .05; Figure 6, B). There was no statistically significant difference in the overall weight of the mice, nor did there appear to be any obvious toxicity from the WIF-1 treatment.


Figure 6
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  		Figure 6. Wnt inhibitory factor (WIF-1) plasmid treatment in an in vivo model using H460 tumor xenografts subcutaneously implanted onto nude mice. Mice underwent peritumoral injections of empty vector or WIF-1. A, Measurements were made at biweekly intervals to determine tumor volume. B, After the mice were killed, tumors were resected and weighed (mean ± standard deviation, P < .05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that restoration of WIF-1 function caused Wnt pathway downregulation and inhibited NSCLC cell growth. Transfection with WIF-1 induced apoptosis in NSCLC cells and inhibited colony formation. Transfection also inhibited NSCLC tumor xenograft growth. Treatment with recombinant WIF-1 protein also inhibited NSCLC proliferation. These results support our previous studies, which showed that inhibition of Wnt signaling could inhibit NSCLC cell growth.19Go The results also reinforce the hypothesis that downregulation of WIF-1 is important for Wnt pathway activation in NSCLC.

Aberrant activation of the Wnt pathway has recently been linked to lung cancer. Traditional alterations in the Wnt pathway, such as ß-catenin and adenomatous polyposis coli gene mutations are relatively uncommon in lung cancer.20-22Go However, there is growing evidence that other mechanisms of Wnt pathway activation do play an important role in lung cancer. We have found evidence of Wnt pathway activation in lung cancer mediated through the molecule disheveled.7Go In addition, multiple studies have shown downregulation of secreted Wnt inhibitors in lung cancer.15,23-25Go WIF-1 is among these secreted Wnt inhibitors and is known to bind Wnt proteins and inhibit their activities.26Go The mechanism of interaction between WIF-1 and Wnt remains poorly understood. Unlike the multiple secreted, frizzled related proteins that have been described, WIF-1 lacks any sequence similarity with the cysteine-rich domain of the Wnt target frizzled receptor.27Go Nonetheless, WIF-1 does bind to Wnt proteins in the extracellular space and inhibits Wnt-frizzled interactions.11Go The relative importance of WIF-1 among the various Wnt antagonists is also unclear.

We have previously shown that WIF-1 is downregulated in the vast majority of NSCLC tumor specimens.15Go The ability of WIF-1 to inhibit NSCLC growth in vitro and in vivo thus has potential therapeutic implications. These experiments reinforce the potential of using demethylating agents to reactivate hypermethylated tumor suppressors in cancer and raise the possibility of therapy specifically related to the role of WIF-1 in the Wnt pathway.28Go


   Footnotes
 
Supported in part by a National Institutes of Health grant (RO1 CA 093708-01A3), the Larry Hall and Zygielbaum Memorial Trust, and the Kazan, McClain, Edises, Abrams, Fernandez, Lyons & Farrise Foundation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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